WO2020132772A1 - Monoclonal antibodies specific for the pb2 antigen of the human influenza virus (flu), nucleotide sequences, method and diagnostic kit for flu infection - Google Patents
Monoclonal antibodies specific for the pb2 antigen of the human influenza virus (flu), nucleotide sequences, method and diagnostic kit for flu infection Download PDFInfo
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Classifications
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- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1018—Orthomyxoviridae, e.g. influenza virus
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- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/54—F(ab')2
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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- C—CHEMISTRY; METALLURGY
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
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- G01N2469/10—Detection of antigens from microorganism in sample from host
Definitions
- Monoclonal antibodies, or fragments thereof, are recognized that recognize the human influenza virus (Flu) virus PB2 protein, where said monoclonal antibodies or fragments thereof comprise an antibody comprising a variable light chain region where its CDR1 (CDR LCI ) is defined according to SEQ ID NO: 1, its CDR2 (CDR LC 2) is defined by SEQ ID NO: 2 and its CDR3 (CDR LC3 ) corresponds to SEQ ID NO: 3, and a variable region of the heavy chain where your CDR1 (CDR HCI ) is defined according to SEQ ID NO: 4, your CDR2 (CDR HC2 ) corresponds to SEQ ID NO: 5 and your CDR3 (CDR HC3 ) corresponds to SEQ ID NO: 6, or an antibody comprising a light chain variable region where its CDR1 (CDR LCI ) is defined according to SEQ ID NO: 7, its CDR2 (CDR LC2 ) is defined by SEQ ID NO: 8 and its CDR3 (CDR LCS
- the present invention relates to monoclonal antibodies, or fragments thereof, that recognize the virus's PB2 protein of human influenza, useful for the development of diagnostic methods of influenza infection in humans.
- Influenza is an infectious contagious disease of the respiratory tract caused by the human influenza virus. This virus is responsible for producing severe or mild respiratory clinical symptoms, mainly affecting the areas of the nose, throat, bronchi and occasionally the lungs. In general, the clinical symptoms of influenza are similar to those of seasonal influenza, however, the symptoms can be variable and range from an asymptomatic infection to severe pneumonia that can cause death 1 . The virus is easily transmitted from person to person through drops or small particles that have been expelled through the cough or sneeze of a sick person, which makes its spread fast and part of seasonal epidemics 2 .
- influenza virus can be detected throughout the year, but its detection is increased in the autumn-winter season, although the time and duration can be variable 3 .
- epidemiological statistics in the United States in 2016, 310,000 people were hospitalized for presenting complications related to influenza. In the same country, statistics indicate that this infection causes around 89,000 deaths annually. From the point of view of economic cost, losses from human influenza viruses in the United States are estimated to reach an annual cost ranging from 71 to 150 billion dollars 4 .
- RIDT rapid influenza diagnostic tests
- RT-PCR reverse transcriptase polymerase chain reaction
- CDC Center for Disease Control and Prevention
- M protein matrix protein
- NP protein nucleoprotein
- NS protein non-structural protein
- Monoclonal antibodies have been previously described for the detection of antigens of the human influenza virus.
- W02012045001 A2
- a human monoclonal antibody is disclosed that binds to the hemagglutinin surface protein.
- a monoclonal antibody or an antigen binding fragment thereof is provided, which can specifically bind to the HA1 domain of the hemagglutinin protein of subtype H1 and subtype H5 of influenza virus.
- the proposed solution aims at the detection of an antigen different from the PB2 protein, and the efficiency, specificity and sensitivity of the antigen-antibody binding in clinical samples are not demonstrated.
- JP2015189715 (A) provides a monoclonal antibody or an antigen-binding fragment thereof that binds to the PB2 subunit of RNA-dependent RNA polymerase.
- JP2015189715 (A) antigen detection tests are not carried out In human clinical samples, the specificity and sensitivity characteristics of the antibodies are not determined in the context of clinical diagnosis. Recall that under clinical diagnostic conditions, biological samples are used that include very low concentrations of antigen, which hinders the specificity and sensitivity of the antigen-antibody reaction.
- a new diagnostic alternative for the human influenza virus is required which, unlike molecular diagnostic tests and cell culture tests that entail longer response times and a high cost for their implementation and maintenance, allows the detection of a wide variety of influenza types and subtypes quickly, sensitively, specifically and at a lower cost. Furthermore, even though monoclonal antibodies have been proposed so far for the detection of other Flu proteins and even against PB2, these antibodies have only been evaluated in murine models and do not correspond in any case to a solution to the technical problem posed.
- monoclonal antibodies that detect the PB2 protein are proposed to be used in the rapid, effective and accurate detection and diagnosis in patients infected with Flu, where said antibodies specifically detect the protein in clinical samples at very low concentrations of the specific antigen (high sensitivity), even distinguishing the specific viral antigen in clinical samples that even include antigens from other respiratory viruses.
- the provided antibodies can be part of a diagnostic method and kit for the diagnosis of Flu, where each of the antibodies It can be used in a versatile way as a detection antibody as a capture antibody.
- the present invention relates to specific monoclonal antibodies against the PB2 protein or fragments thereof, of the human influenza virus.
- the invention corresponds to monoclonal antibodies or fragments thereof secreted by hybridoma cell lines called 1A3E2 and 2F11B1, which recognize the PB2 protein of the human influenza virus (Flu), where said monoclonal antibodies or fragments of these comprise an an antibody comprising a light chain variable region where its CDR1 (CDR LCI ) is defined by SEQ ID NO: 1, its CDR2 (CDR LC2 ) is defined by SEQ ID NO: 2 and its CDR3 (CDR LCS ) corresponds to SEQ ID NO: 3, and a variable region of the heavy chain where your CDR1 (CDR HCI ) is defined according to SEQ ID NO: 4, your CDR2 (CDR HC2 ) corresponds to
- SEQ ID NO: 5 and its CDR3 corresponds to SEQ ID NO: 6, or an antibody that comprises a light chain variable region where its CDR1 (CDR LCI ) is defined according to SEQ ID NO: 7, its CDR2 (CDR LC2) is defined by SEQ ID NO: 8 and its CDR3 (CDR LCS ) corresponds to SEQ ID NO: 9, and a variable region of the heavy chain where its CDR1 (CDR HCI ) is defined according to SEQ ID NO: 10, its CDR2 (CDR HC2) corresponds to SEQ ID NO: 11 and its CDR3 (CDR HCS ) corresponds to SEQ ID NO: 12, where the antibody can be used as detection antibody or capture antibody.
- a method of diagnosing Flu infection in a biological sample uses the monoclonal antibodies in diagnostic kit format to detect Flu, where said kit comprises at least one monoclonal antibody against Flu as previously described.
- the antibodies described in the invention present important advantageous and remarkable technical characteristics with respect to other antibodies and detection methods of viral antigens that already exist.
- each virus has specific surface proteins, therefore, other diagnostic techniques based on monoclonal antibodies for other types of respiratory viruses are not comparable to the proposed invention.
- the detection specificity of the antibodies against the Flu PB2 protein relative to other viral antigens, for example against the adenovirus pIII protein, is demonstrated in the results provided in Figures 1A, IB and 1C. From these results it is possible to conclude that the antibodies provided in the scope of the present application only recognize Flu's PB2 protein, and that in ELISA tests with ADV virus antigens no detection signal was observed.
- the antibodies that are part of the scope of the invention make it possible to specifically detect the PB2 protein or fragments thereof, so that they do not compete with each other for the antigen binding site, nor do they impede their simultaneous binding to East.
- PB2 protein or fragments of it allow the detection of the PB2 protein or fragments of it with high sensitivity in samples that contain a low amount of antigen, such as nasopharyngeal swab samples, for example.
- the proposed monoclonal antibodies are capable of detecting PB2 protein, a highly conserved protein.
- the detection strategy of a conserved viral protein allows antibodies that are within the scope of the invention to detect different types of human influenza, including influenza A, B, and C.
- CDR sequences When CDR sequences are referred to in the present invention, they correspond to short sequences that are found in the variable domains of proteins that have antigen detection function.
- the CDR sequences for heavy chain (CDR HC ) and light chain (CDRi, c) of the antibodies secreted by 1A3E2 and 2F11B1 hybridomas are presented.
- the described monoclonal antibodies can be used for detection, diagnosis and / or determination of Flu infection. These antibodies can be used simultaneously to increase detection sensitivity in clinical samples where there is little quantity and availability of antigen.
- a method of diagnosing Flu infection in a biological sample which comprises contacting the biological sample with the monoclonal antibody against Flu PB2 protein or a fragment thereof according to claim, and detect binding of the antibody to the antigen.
- the biological sample may correspond, without limitation, to in vitro cells infected with Flu, nasal secretions, nasal lavages, cerebrospinal fluid, pharyngeal secretions and / or bronchial lavages or secretions.
- the assay used for detection of antigen-antibody binding is selected from ELISA, Luminex, immunofluorescence, immunohistochemistry, immunochromatography, flow cytometry, cell sorter, immunoprecipitation, and / or Western blot analysis.
- the present invention also includes a diagnostic kit for detecting human influenza virus, which comprises: a monoclonal antibody against Flu PB2 protein or a fragment thereof, where said antibody can act as a capture or detection antibody , where particularly, the detection antibody is conjugated to a marker for detection; a solid support to which the antibody is attached; and reagents for detecting the marker included in the detection antibody, such as fluorophores, biotin, radioisotopes, metals, and enzymes.
- a diagnostic kit for detecting human influenza virus which comprises: a monoclonal antibody against Flu PB2 protein or a fragment thereof, where said antibody can act as a capture or detection antibody , where particularly, the detection antibody is conjugated to a marker for detection; a solid support to which the antibody is attached; and reagents for detecting the marker included in the detection antibody, such as fluorophores, biotin, radioisotopes, metals, and enzymes.
- capture antibody when referring to capture antibody, this corresponds to the antibody that specifically binds to the antigen.
- detection antibody this corresponds to the antibody to which a marker is conjugated to be detected by different tests such as immunochromatographic test, luminex, flow cytometry, immunofluorescence, radioimmunoassay, Western blot, Dot plot, ELISA, luminex, immunodiffusion. or immunoprecipitation.
- the antibodies part of the present invention can dual function as a capture antibody or as a detection antibody when coupled to the detection marker.
- the detection marker will be conjugated to the detection antibody, and this can correspond, without limitation, to fluorophores, biotin, radioisotopes, metals and enzymes.
- the detection antibody is conjugated to the reporter system based on detection of activity of horseradish peroxidase enzyme (HRP).
- HRP horseradish peroxidase enzyme
- Figure 1 Detection of Flu PB2 protein by monoclonal antibodies produced by hybridomas 1A3E2 and 2F11B1, by means of an indirect ELISA assay.
- the plate was activated with 50 ng of purified recombinant Flu PB2 protein, 50 ng of ADV pIII protein (as a specificity control) and 20 pg of uninfected MDCK cells (used as a specificity control) and infected with Flu. Control wells without antigen, with primary antibody, with anti-IgG were included. mouse conjugated to HRP (not activated) and wells without antigen or primary antibody, only with anti-mouse IgG antibody (HRP), data not shown in the graph.
- HRP not activated
- the wells were incubated with the anti-PB2 antibodies from the 1A3E2 hybridoma, in the amount of 170 ng (A), the 2F11B1 hybridoma in the amount of 170 ng (B) and the commercial polyclonal antibody Anti-Influenza A virus PB2 protein antibody , catalog number GTX125926 (GeneTex) used in quantity of 170 ng (C).
- the data shown in the graph expresses the absorbance (in OD, optical density) detected at 450 nm, emitted by the conversion of the substrate Tetramethylbenzidine to a colored compound, catalyzed by the enzyme Horseradish peroxidase (HRP) conjugated in a secondary anti-IgG antibody mouse that specifically bound to the antibodies secreted by GeneTex hybridomas 1A3E2, 4D8C6 and GTX125926. Values correspond to the average +/- standard deviation of absorbance emitted by each sample in at least two independent experiments.
- HRP horseradish peroxidase
- Figure 2 Determination of sensitivity of monoclonal antibodies produced by 1A3E2 and 2F11B1 hybridomas in the detection of Flu PB2 protein.
- ELISA plates were activated with 1: 2 serial dilutions, starting with 50 ng of PB2 protein and ending with 0.04 ng. Subsequently, the wells were incubated with the anti-PB2 antibodies from the 1A3E2 hybridoma, in the amount of 170 ng (A) and the 2F11B1 hybridoma in the amount of 170 ng (B). Unactivated wells were included as a negative control.
- the data shown in the graph express the absorbance at 450 nm emitted by the conversion of the substrate Tetramethylbenzidine to a colored compound catalyzed by the Horseradish peroxidase (HRP) enzyme conjugated to anti-PB2 antibodies from hybridomas 1A3E2 and 2F11B1 in an amount of 170 ng (A and B). Values correspond to the average +/- standard deviation of absorbance emitted by each sample in at least two independent experiments. * P ⁇ 0.05; ** P ⁇ 0.01; *** P ⁇ 0.001 by the parametric student test comparing the results of the well called control versus each of the dilutions of the PB2 protein.
- HRP Horseradish peroxidase
- Figure 3 Assay of serial dilutions of Flu anti-PB2 monoclonal antibodies produced by 1A3E2 and 2F11B1 hybridomas, for the detection of purified Flu antigens.
- ELISA plates were activated with 50 ng of recombinant Flu PB2 protein and the antigen was detected with 11 serial dilutions 1: 2 of the anti-PB2 antibodies 1A3E2 (A) or 2F11B1 (B), starting from a concentration of 3.4 mr / hIE (170 ng per well).
- Data are expressed as the average +/- standard deviation of the absorbance value emitted at 450 nm for each sample in duplicate, in at least two independent experiments.
- Figure 4 Detection of Flu in clinical samples by sandwich ELISA, using the combination of monoclonal antibodies secreted by hybridomas 1A3E2 and 2F11B1.
- ELISA plates were activated with 170 ng of antibody secreted by the 1A3E2 hybridoma (anti-Flu), functioning as a capture antibody.
- the wells activated with the capture antibody were incubated with 50 m ⁇ of nasopharyngeal swab (HNF) samples from patients presenting with viral respiratory symptoms.
- HNF nasopharyngeal swab
- As negative controls 10 samples from healthy controls. Twelve samples from Flu-positive patients were used and as a specificity control, 3 samples from patients positive for Parainfluenza virus were included.
- wells were included to which purified recombinant PB2-Flu protein was added.
- the antibodies produced by the 2F11B1 hybridoma, conjugated to the Horseradish Peroxidase enzyme were used in a 1: 2000 dilution (1.8 ng / m ⁇ per well).
- the data shown is the median value of the absorbance emitted at 450 nm for each sample (** P ⁇ 0.01 **** and p ⁇ 0.0001; using the non-parametric student test and the Mann Whitney post test comparing Flu positive patients versus healthy controls, and against the viruses used as specificity control).
- Figure 5 Detection of PB2 protein by indirect ELISA assay, using whole monoclonal antibodies and fragments of them, secreted by biotin-conjugated 1A3E2 and 2F11B1 hybridomas. Detection of PB2 protein from Biotin-conjugated antibodies is observed. Fragments of the antibody secreted by hybridomas 1A3E2 and 2F11B1 respectively are indicated in black and white. While in gray the activity of the complete fragments of the antibodies secreted by the 1A3E2 and 2F11B1 hybridomas respectively is presented.
- the data shown in the graph expresses the absorbance at 450 nm emitted by the conversion of the substrate Tetramethylbenzidine to a colored compound catalyzed by the enzyme Horseradish peroxidase (HRP).
- HRP Horseradish peroxidase
- the average of the absorbance value emitted at 450 nm of each sample is shown (where b is equal to p ⁇ 0.0001 compared to a; by means of the 2-way ANOVA test comparing the well without sample versus the well with protein with all antibodies). Examples that demonstrate the different applications of the monoclonal antibodies of the invention.
- Example 1 Determination of the nucleotide sequence encoding the light (VL) and heavy (VH) chains of the variable region of the anti PB2 Flu antibody secreted by the 1A3E2 hybridoma.
- the 1A3E2 hybridoma was grown in DMEM-high glucose culture medium supplemented with 3.7 g / L Sodium Bicarbonate and 10% fetal bovine serum, at 37 ° C with 10% CO2, up to a cell density of 700,000 cells / mL.
- the total RNA of 3.5 x 10 cells was obtained, carrying out a treatment with the compound Trizol (Invitrogen).
- 0.5 mg of RNA was used to generate the cDNA by reverse transcription reaction with the PrimeScriptTM lst Strand cDNA Synthesis Kit, which uses isotype specific universal cleavers.
- the light and heavy chain of the antibody were amplified according to the GenScript cDNA Extreme Rapid Amplification (RACE) Standard Operating Procedure (SOP).
- RACE GenScript cDNA Extreme Rapid Amplification
- SOP Standard Operating Procedure
- Amplified antibody fragments were cloned separately into a standard cloning vector. Colony PCR was performed to identify clones that had inserts of the correct size. At least five colonies with correct size inserts were sequenced for each fragment. The sequences of different clones were aligned and the consensus sequence of these clones was provided. The nucleotide sequences of the heavy and light chains of the antibodies secreted by the 1A3E2 hybridoma were identified, being identified with SEQ ID NO. 1 and SEQ ID NO.3 for the case of heavy chains and SEQ ID NO. 2 and SEQ ID NO.4 in the case of light chains.
- Example 2 Determination of the nucleotide sequence encoding the light (VL) and heavy (VH) chains of the variable region of the anti-PB2 Flu antibody secreted by the 2F11B1 hybridoma.
- the 2F11B1 hybridoma was grown in DMEM-high glucose culture medium supplemented with 3.7 g / L Sodium Bicarbonate and 10% fetal bovine serum, at 37 ° C with 10% CO2, up to a cell density of 700,000 cells / mL.
- the total RNA of 3.5 x 10 cells was obtained, carrying out a treatment with the compound Trizol (Invitrogen).
- 0.5 mg of RNA was used to generate the cDNA by reverse transcription reaction with the PrimeScriptTM lst Strand cDNA Synthesis Kit, which uses isotype specific universal cleavers.
- the light and heavy chain of the antibody were amplified according to the GenScript cDNA Extreme Rapid Amplification (RACE) Standard Operating Procedure (SOP).
- Amplified antibody fragments were cloned separately into a standard cloning vector. Colony PCR was performed to identify clones that had inserts of the correct size. At least five colonies with correct size inserts were sequenced for each fragment. The sequences of different clones were aligned and the consensus sequence of these clones was provided. From this, the nucleotide sequences of the heavy and light chains of the antibodies secreted by the 2F11B1 hybridoma were determined, corresponding to the sequences identified as SEQ ID NO. 1 and SEQ ID NO .3 to the light chains and the sequences identified as SEQ ID NO. 1 and SEQ ID NO.3 to heavy chains.
- Example 3 Flu antigen detection assay, determination of specificity of Flu anti-PB2 monoclonal antibodies for purified Flu antigens by indirect ELISA assay.
- This assay aims to demonstrate the specificity for Flu PB2 protein of the antibodies produced by hybridomas 1A3E2 and 2F11B1.
- Antigen detection was carried out using the indirect ELISA technique, where the ELISA plate was activated with 50 ng of purified antigen for 1 hour at 37 ° C. Likewise, the plate was activated with 20 pg of Cell used from uninfected MDCK cells (as a negative control) and infected with Flu serotype A virus. Another negative control included was 50 ng of ADV pIII protein in a separate well. Subsequently, the plate was washed twice with phosphate buffered saline (PBS) lX / Tween20 0.05%.
- PBS phosphate buffered saline
- the plate was then blocked for 2 hours at 37 ° C with PBS IX / 10% Fetal Bovine Serum (FBS). Subsequently, the washes were repeated and each of the antibodies (1A3E2 and 2F11B1) were then incubated at a final concentration of 3.4 pg / mL (170 ng per well), diluted in PBS 1X / FBS 10%, for 1 hour at 37 ° C (each antibody on a separate plate). Under the same conditions, on a different plate, a control assay was performed using a commercial monoclonal antibody that recognizes Flu's PB2 protein (catalog number GTX125926, GeneTex) at a concentration of 3.4. pg / mL.
- FBS Fetal Bovine Serum
- Example 4 Assay to determine the sensitivity of monoclonal antibodies for the detection of Flu anti-PB2 viral antigens.
- the assay was performed to determine the maximum protein dilution that Flu anti-PB2 monoclonal antibodies from 1A3E2 and 2F11B1 hybridomas are capable of detecting by indirect ELISA.
- the plate was activated with 11 serial dilutions 1: 2 of Flu PB2 protein, starting with 50 ng of purified antigen.
- Anti-PB2 1A3E2 or 2F11B1 antibodies were used at a concentration of 3.4 pg / mL (170 ng / well), diluted in 1X PBS / 10% FBS.
- the anti-mouse IgG detection antibody was added at a dilution of 1: 2,000 (1.8 ng / m ⁇ per well) and incubated 1 hour at room temperature (25 ° C), in the dark. Finally, washes were performed and revealed with 50 pL of citrate / Tetramethylbenzidine buffer (TMB, 3-3 '-5-5' tetramethylbenzidine, lmg / mL, Becton Dickinson). To stop the reaction, 50 pL of H2SO4 2N were added and the result was read in an ELISA reader, at 450nm.
- TMB citrate / Tetramethylbenzidine buffer
- the anti-PB2 antibody 1A3E2 is capable of recognizing up to 780 picograms (pg) of the Flu2 PB2 protein ( Figure 2A).
- the anti-PB2 antibody from the 2F11B1 hybridoma showed the same sensitivity as the anti-PB2 1A3E2 antibody ( Figure 2B). Controls that allowed to rule out nonspecific reactions of both the antibodies, which contained all components of the assay except the sample (PB2 Flu protein, data not shown in the graphs) were included in all the tests.
- Example 5 Assay to determine the efficiency of monoclonal antibodies to detect Flu viral antigens, by indirect ELISA.
- the assay was performed to determine the maximum dilution of Flu's anti-PB2 monoclonal antibodies from 1A3E2 and 2F11B1 hybridomas, which allow detection of antigen viral.
- the plate was activated with 50 ng of purified antigen (PB2 protein) and then the plate was blocked for 2 hours at 37 ° C with PBS IX / 10% Fetal Bovine Serum (FBS).
- Anti-PB2 1A3E2 or 2F11B1 antibodies were used in 1: 2 dilutions, starting from the working concentration (170 ng) up to the 11 dilution (0.15 ng) in PBS 1X / 10% FBS.
- the anti-mouse IgG detection antibody was added at a dilution of 1: 2000 (1.8 ng / m ⁇ per well) and it was incubated for 1 hour at room temperature (25 ° C), in the dark. Finally, the washes were carried out and it was developed with 50 pL of citrate / Tetramethylbenzidine buffer (TMB, 3-3 '-5-5' tetramethyl-benzidine, lmg / mL, Becton Dickinson). To stop the reaction, 50 pL of H2SO4 2N were added and the result was read in an ELISA reader, at 450 nm.
- TMB citrate / Tetramethylbenzidine buffer
- Figure 3 shows that the anti-PB2 antibody 1A3E2 can detect 50 ng of the purified antigen with up to 1.3 ng per well (Figure 3A).
- the anti-PB2 clone 2F11B1 is more efficient than the clone 1A3E2, since it recognizes 50 ng of purified PB2 with almost all the dilutions made ( Figure 3B).
- the negative control included in this assay corresponds to a well that does not contain a sample (PB2 protein), was blocked with PBS 1X / FBS 10%, primary antibody was added (anti-PB2 1A3E2 or anti-PB2 2F11B1) and also contains HRP-conjugated anti-mouse IgG antibody.
- Example 6 Clinical diagnosis of samples from patients infected with Flu, using Flu anti-PB2 monoclonal antibodies, using the sandwich ELISA technique.
- wells of an ELISA plate were activated with 3.4 mr / hIII (170 ng / well) of the anti-PB2 antibody from the 1A3E2 hybridoma for Flu, which was diluted in PBS IX, and incubated for 1 hour at 37 ° C. 2 washes were performed with 0.05% PBS lX-Tween20 and the plate was subsequently blocked with 200 mE of 1X PBS / 10% FBS for 1 hour at 37 ° C.
- Figure 4A The results obtained for this assay are shown in Figure 4A, where it can be seen that the sandwich ELISA technique using the antibody (anti-PB2) from the 1A3E2 hybridoma, as the capture antibody and the antibody from the 2F11B1-HRP hybridoma As a detection antibody, it allows the antigen to be detected in samples from patients infected with Flu ( Figure 4A), which were previously confirmed by direct immunofluorescence in a certified clinical laboratory using the viral panel.
- Figure 4A shows the results obtained with the Flu anti-PB2 antibodies, where 12 samples from patients diagnosed as Flu positive were used and as a specificity control, 3 samples from patients positive for the Parainfluenza virus were included.
- PB2-Flu protein As a positive control, wells were included to which purified recombinant PB2-Flu protein was added. As a negative control, 10 healthy controls were used. The results show that the antibodies are specific to detect only Flu-positive patients and not healthy controls or those infected with another virus (PIV). All samples detected positive by ELISA are those showing an optical density (OD) above 0.15.
- This assay demonstrates the versatility of the antibodies from Flu anti-PB2 hybridomas 1A3E2 and 2F11B1, since they are capable of simultaneously binding to the antigen without competing for the binding site or interfering with each other. This allows the capture and subsequent detection of PB2 protein in patient samples.
- the samples used for the tests were obtained from nasopharyngeal swabs contained in universal transport medium (UTM). Samples were centrifuged at 14,000 rpm for 4 minutes at room temperature. The supernatant was subsequently separated (SN1) of the pellet; the latter was incubated with 100 m ⁇ of RIPA Buffer (50 M Tris-HCl pH 8.0, 150 M NaCl, 1% NP-40, 0.5% Sodium Deoxycholate, 0.1%, SDS and a cocktail of inhibitors of proteases IX) for 15 minutes at 4 ° C, vortexing every 5 minutes. Then, it was spun at 14,000 rpm for 4 minutes at room temperature. At the end, the obtained supernatant (SN2) was taken and mixed with SN1, a vortex was performed.
- RIPA Buffer 50 M Tris-HCl pH 8.0, 150 M NaCl, 1% NP-40, 0.5% Sodium Deoxycholate, 0.1%, SDS and a cocktail of inhibitors of proteases IX
- Example 7 Clinical diagnosis of samples from patients infected with FLU, using anti-FLU monoclonal antibodies to FLU, using the Sandwich-type Luminex technique.
- the availability and concentration of viral proteins is generally very low in clinical samples of nasopharyngeal swabs, so we wanted to evaluate by another more sensitive technique what was obtained in the results by the ELISA technique.
- Figure 4A For this assay, a Sandiwch-type luminex assay was performed, using the anti-PB2 1A3E2 antibody as the capture antibody and the anti-PB2 2F11B1 as the detection antibody.
- the FLU anti-PB2 detection antibody 2F11B1 was conjugated to the biotin fluorophore.
- Luminex plates were activated with 50 magnetic microspheres (internally marked with red or near-infrared fluorophore of different intensities) by m ⁇ , which were conjugated with the antibody secreted by the 1A3E2 hybridoma (anti-FLU), functioning as a capture antibody ( at a final concentration of 2.5 mM).
- the conjugated microspheres were incubated with 50 m ⁇ of nasopharyngeal swab (HNF) samples from patients presenting with viral respiratory symptoms, for 2 hours at room temperature ("23 ° C), shaking at 400 rpm and in the dark (covered with tissue paper). aluminum) .
- HNF nasopharyngeal swab
- Incubation is carried out for 1 hour at room temperature, in the dark, shaking at 400 rpm. 2 washes are again performed with 100 mB 0.05% PBS ⁇ X-Tween20 for 30 seconds using the manual magnetic scrubber. The complex formed by microspheres conjugated with capture antibody plus antigen and detection antibody, is incubated with 50 mE of Streptavidin / Fi-coeritrin at a final concentration of 6 mr / h ⁇ . Incubation is carried out for 30 minutes at room temperature, in the dark, shaking at 400 rpm.
- FIG. 4B shows the results obtained for this assay.
- the Luminex technique like that obtained by the ELISA technique, using the antibody (anti-PB2) from the 1A3E2 hybridoma, as a capture and the antibody from the 2F11B1-HRP hybridoma as a detection antibody, allows the antigen to be detected in samples from patients infected with FLU ( Figure 4A) with high intensity, which were previously confirmed by direct immunofluorescence in a certified clinical laboratory using the panel viral.
- Figure 4B shows the results obtained with the FLU anti-PB2 antibodies, where 19 samples from patients diagnosed as positive FLU and 6 samples from healthy controls were used. In addition, wells to which purified PB2-FLU protein was added were used as a positive control.
- the results show that anti-PB2 antibodies are specific in detecting only FLU positive patients and not control subjects. All samples detected as positive by Luminex are those showing an MFI above two standard deviations from the average MFI of healthy controls.
- This assay demonstrates the versatility of antibodies from FLU 1A3E2 and 2F11B1 hybridomas, since they are capable of simultaneously binding to the antigen without competing for the binding site or interfere with each other and detect the low availability of the antigen in the nasopharyngeal swab sample.
- Example 8 Blind study for the detection of the PB2-FLU antigen in clinical samples, obtained from patients carrying an infection, using anti-FLU monoclonal antibodies to FLU, which are part of the multiple respiratory virus detection kit.
- sandwich ELISA tests were performed where the previous diagnosis of the samples to be evaluated was known. Subsequent to these trials, a blind study was performed, where nearly 160 samples of nasopharyngeal swabs were evaluated, without knowing the microbiological diagnosis.
- Sandwich ELISAs were performed where the anti-L 1A3E2 antibody and the anti-L 2F11B1 were used as the HRP-conjugated detection antibody. For all tests, wells of a plate were activated. ELISA with 3.4 mg / mL (170 ng / well) of the anti-L antibody from the FLU 1A3E2 hybridoma, diluted in PBS IX, for 30 minutes at 37 ° C.
- the results are shown in figure 4A, where the ability of the antibodies to detect the PB2 protein in clinical samples is observed, since they were designed from a chimera protein. 18 out of 21 positive IVP patients were detected, and from these results the diagnostic accuracy of the antibodies could be determined, which is shown in Table 1.
- Table 1 shows the two concepts that define the diagnostic accuracy, where we have specificity, that is, the ability of antibodies to diagnose negative as negative samples, without detecting false positives, and on the other hand, we have sensitivity, that is, the ability of antibodies to diagnose as positive those samples that They really are, without diagnosing false negatives.
- the results shown in the table show a high percentage of specificity (94%) and sensitivity (86%) of the antibodies against the standard technique (PCR).
- Example 9 Detection of PB2 protein by indirect ELISA assay, using whole monoclonal antibodies and fragments of them.
- both the specific monoclonal antibody against the PB2 protein can be detected by indirect ELISA.
- ELISA plates were activated with 50 mE of 50 ng of PB2 protein and BSA. Non-specific sites were blocked with 10% FBS diluted in PBS IX. 170 ng (3.4 mg / mL) of the Fab fragments of the antibodies secreted by the hybridoma 1A3E2 (anti-Flu) and 2F11B1 (anti-Flu), both previously conjugated biotin.
- biotin-binding molecules Streptavidin
- HRP 1-: 2000 dilution, 75 ng per well
- Example 10 Flu antigen detection assay, using F (ab ') 2 fragments of Flu anti-PB2 monoclonal antibodies by indirect ELISA assay
- This assay aims to demonstrate the ability to detect fragments of anti-Flu antibodies, produced by hybridomas 1A3E2 and 2F11B1, by protein PB2.
- the IgG molecule of each anti-Flu antibody was fragmented. Fragmentation was performed using the "Thermo Scientific TM F (ab ') 2 Pierce TM Fragment Preparation Kits" kit (# 10381214, Thermo Scientific), which separates the F (ab') 2 fragment and Fe from the Of interest, by using the enzyme pepsin that digests the Fe fragment and subsequently purification steps are performed to separate the F (ab ') 2 fragment from the digested Fe fragment.
- F (ab ') 2 fraction was verified by the western blot technique.
- F (ab ') 2 fractions were conjugated to biotin molecules using the Lightning-Link rapid biotin type A rapid conjugation kit (# 370-0010, Expedeon). Having all the reagents ready, the detection of the antigen was carried out using the indirect ELISA technique, where the ELISA plate was activated with 50 ng of purified PB2 antigen for 1 hour at 37 ° C. Two negative controls were included, one without a sample and the other incubating the well with 50 ng of BSA protein.
- the plate was washed twice with phosphate buffered saline (PBS) lX / Tween20 0.05%. The plate was then blocked for 2 hours at 37 ° C with PBS IX / 10% Fetal Bovine Serum (FBS). Subsequently, the washes were repeated and each of the antibodies was then incubated, without fractionating and biotin-conjugated F (ab ') 2 fractions (1A3E2 and 2F11B1), at a final concentration of 3.4 mg / mL (170 ng per well), diluted in PBS 1X / FBS 10%, for 1 hr at 37 ° C (each antibody on a separate plate).
- PBS phosphate buffered saline
- FBS Fetal Bovine Serum
- the washes were repeated and a biotin-binding protein (Streptavidin) labeled with the horseradish peroxidase enzyme (Horseradish peroxidase, HRP) in dilution 1 in 2000 (25 ng) was added to each well. / mE per well) in PBS 1X / FBS 10%, for 1 hour at room temperature (25 ° C), in the dark. Finally, the washes were performed and revealed with 50 L of citrate / Tetramethylbenzidine buffer (TMB, 3-3 '-5- 5' tetramethylbenzidine, lmg / ml, Becton Dickinson).
- TMB citrate / Tetramethylbenzidine buffer
Abstract
The invention relates to the generation of monoclonal antibodies, or fragments of same, which recognises the PB2 protein of the human influenza virus (flu), wherein the monoclonal antibodies or fragments of same comprise a variable domain of the heavy chain and a variable domain of the light chain. Also provided is a diagnostic method for detecting flu infections in biological samples of nasopharyngeal secretions, using the monoclonal antibodies in the format of a diagnostic kit.
Description
ANTICUERPOS MONOCLONALES ESPECÍFICOS PARA EL ANTÍGENO PB2 DEL VIRUS DE LA INFLUENZA HUMANA (FLU) , SECUENCIAS NUCLEOTÍDICAS ; MÉTODO Y KIT DE DIAGNÓSTICO DE INFECCIÓN PRODUCIDA POR FLUSPECIFIC MONOCLONAL ANTIBODIES FOR THE PB2 ANTIGEN OF HUMAN INFLUENZA VIRUS (FLU), NUCLEOTIDIC SEQUENCES; DIAGNOSTIC METHOD AND KIT OF INFECTION PRODUCED BY FLU
MEMORIA DESCRIPTIVA Descripción de la invención DESCRIPTIVE MEMORY Description of the invention
Se presentan anticuerpos monoclonales , o fragmentos de los mismos que reconocen la proteina PB2 del virus de la influenza humana (Flu), donde dichos anticuerpos monoclonales o fragmentos de estos comprenden una un anticuerpo que comprende una región variable de cadena ligera donde su CDR1 (CDRLCI) se define según la SEQ ID NO: 1, su CDR2 (CDRLC2) se define por la SEQ ID NO: 2 y su CDR3 (CDRLC3) corresponde a la SEQ ID NO: 3, y una región variable de la cadena pesada donde su CDR1 (CDRHCI) se define según la SEQ ID NO: 4, su CDR2(CDRHC2) corresponde a la SEQ ID NO: 5 y su CDR3 (CDRHC3) corresponde a la SEQ ID NO: 6, o un anticuerpo que comprende una región variable de cadena ligera donde su CDR1 (CDRLCI) se define según la SEQ ID NO: 7, su CDR2 ( CDRLC2 ) se define por la SEQ ID NO: 8 y su CDR3 (CDRLCS) corresponde a la SEQ ID NO: 9, y una región variable de la cadena pesada donde su CDR1 (CDRHCI) se define según la SEQ ID NO: 10, su CDR2 (CDRHC2 ) corresponde a la SEQ ID NO: 11 y su CDR3 (CDRHCS) corresponde a la SEQ ID NO: 12, donde el anticuerpo puede ser usado como anticuerpo de detección o anticuerpo de captura. Adicionalmente, se proporciona un método de diagnóstico de infección por Flu en una muestra biológica que utiliza los anticuerpos monoclonales en formato de kit de diagnóstico para detectar Flu, donde dicho kit comprende al menos un anticuerpo monoclonal contra Flu de acuerdo a lo descrito previamente. Monoclonal antibodies, or fragments thereof, are recognized that recognize the human influenza virus (Flu) virus PB2 protein, where said monoclonal antibodies or fragments thereof comprise an antibody comprising a variable light chain region where its CDR1 (CDR LCI ) is defined according to SEQ ID NO: 1, its CDR2 (CDR LC 2) is defined by SEQ ID NO: 2 and its CDR3 (CDR LC3 ) corresponds to SEQ ID NO: 3, and a variable region of the heavy chain where your CDR1 (CDR HCI ) is defined according to SEQ ID NO: 4, your CDR2 (CDR HC2 ) corresponds to SEQ ID NO: 5 and your CDR3 (CDR HC3 ) corresponds to SEQ ID NO: 6, or an antibody comprising a light chain variable region where its CDR1 (CDR LCI ) is defined according to SEQ ID NO: 7, its CDR2 (CDR LC2 ) is defined by SEQ ID NO: 8 and its CDR3 (CDR LCS ) corresponds to SEQ ID NO: 9, and a variable region of the heavy chain where your CDR1 (CDR HCI ) is defined according to SEQ ID NO: 10, your CDR2 (CDR HC2 ) corresponds to the SEQ ID NO: 11 and its CDR3 (CDR HCS ) corresponds to SEQ ID NO: 12, where the antibody can be used as detection antibody or capture antibody. Additionally, a method of diagnosing Flu infection in a biological sample is provided that uses the monoclonal antibodies in diagnostic kit format to detect Flu, where said kit comprises at least one monoclonal antibody against Flu as previously described.
CAMPO DE LA INVENCIÓN FIELD OF THE INVENTION
La presente invención se refiere a anticuerpos monoclonales, o fragmentos de los mismos, que reconocen la proteina PB2 del virus
de la influenza humana, útil para el desarrollo de métodos de diagnóstico de infección de influenza en humanos . The present invention relates to monoclonal antibodies, or fragments thereof, that recognize the virus's PB2 protein of human influenza, useful for the development of diagnostic methods of influenza infection in humans.
ANTECEDENTES DE LA INVENCIÓN BACKGROUND OF THE INVENTION
La influenza, es una enfermedad infectocontagiosa de las vías respiratorias provocada por el virus de la influenza humana. Este virus es responsable de producir cuadros clínicos respiratorios graves o leves, afectando principalmente las zonas de la nariz, garganta, bronquios y ocasionalmente los pulmones. En general, los sintomas clínicos de la influenza son parecidos a los de una gripe estacional, sin embargo, los sintomas pueden ser variables y van desde una infección asintomática hasta una neumonía grave que puede provocar la muerte1. El virus se transmite con facilidad de persona a persona a través de gotas o pequeñas partículas que han sido expulsadas a través de la tos o estornudos de una persona enferma, lo cual hace que su propagación sea rápida y forme parte de epidemias estacionales2. Influenza is an infectious contagious disease of the respiratory tract caused by the human influenza virus. This virus is responsible for producing severe or mild respiratory clinical symptoms, mainly affecting the areas of the nose, throat, bronchi and occasionally the lungs. In general, the clinical symptoms of influenza are similar to those of seasonal influenza, however, the symptoms can be variable and range from an asymptomatic infection to severe pneumonia that can cause death 1 . The virus is easily transmitted from person to person through drops or small particles that have been expelled through the cough or sneeze of a sick person, which makes its spread fast and part of seasonal epidemics 2 .
El virus de la influenza puede ser detectado durante todo el año, pero su detección se incrementa en la temporada otoño- invierno, aunque la época y la duración puede ser variable3. Según estadísticas epidemiológicas, en Estados Unidos en el año 2016, 310.000 personas fueron hospitalizadas por presentar complicaciones relacionadas con influenza. En el mismo pais, la estadística indica que esta infección provoca alrededor de 89.000 muertes anuales. Desde el punto de vista del costo económico, las pérdidas por virus de influenza humana en Estados Unidos se estima alcanzan un costo anual que va desde los 71 a 150 mil millones de dólares4. The influenza virus can be detected throughout the year, but its detection is increased in the autumn-winter season, although the time and duration can be variable 3 . According to epidemiological statistics, in the United States in 2016, 310,000 people were hospitalized for presenting complications related to influenza. In the same country, statistics indicate that this infection causes around 89,000 deaths annually. From the point of view of economic cost, losses from human influenza viruses in the United States are estimated to reach an annual cost ranging from 71 to 150 billion dollars 4 .
1 1. http://www.who.int/csr/disease/swineflu/faq/es/tdoesit 1 1. http://www.who.int/csr/disease/swineflu/faq/es/tdoesit
2 http://www.paho.org/hq/¡ndex.php?opt¡on=com_content&v¡ew=art¡cle&¡d=3154<em¡d=2498&lang=es 2 http://www.paho.org/hq/¡ndex.php?opt¡on=com_content&v¡ew=art¡cle&¡d=3154<em¡d=2498&lang=es
3 https://espanol.cdc.gov/enes/flu/about/season/flu-season.htm 3 https://espanol.cdc.gov/enes/flu/about/season/flu-season.htm
4 https://espanol.cdc.gov/enes/flu/about/disease/us_flu-related_deaths.htm
Los métodos de diagnóstico más comunes para la detección de Flu se denominan "pruebas de diagnóstico rápido de influenza" (RIDT) , estas pruebas se basan en la detección de antigenos de Flu ( inmunoensayo ) en muestras de hisopado o aspirado nasofaríngeo. Estas pruebas pueden entregar un resultado en un periodo de 15 a 20 minutos, sin embargo, carecen de sensibilidad y solo confieren un resultado cualitativo (positivo o negativo), que puede ser potencialmente un falso negativo debido a su baja especificidad5. 4 https://espanol.cdc.gov/enes/flu/about/disease/us_flu-related_deaths.htm The most common diagnostic methods for detecting Flu are called "rapid influenza diagnostic tests" (RIDT), these tests are based on the detection of Flu antigens (immunoassay) in swab or nasopharyngeal aspirate samples. These tests can deliver a result in a period of 15 to 20 minutes, however, they lack sensitivity and only confer a qualitative result (positive or negative), which can potentially be a false negative due to its low specificity. 5
Hasta ahora, la técnica estándar para la confirmación de una infección por el virus de la influenza corresponde al análisis molecular de la reacción en cadena de la polimerasa con transcriptasa inversa (RT-PCR) . Por ejemplo, el Panel de Diagnóstico de RT-PCR en Tiempo Real del Virus de Influenza Humana desarrollado por el Centro para el Control y Prevención de Enfermedades (CDC, por sus siglas en inglés) permite la detección ín vítro del virus de la influenza en muestras de vias respiratorias de pacientes humanos que presentan signos y sintomas de infección respiratoria. Este método detecta los virus de influenza A y B a través de la reacción de partidores contra genes codificantes de proteínas altamente conservadas, tales como la proteina de la matriz (proteina M) , nucleoproteina (proteina NP) y proteina no estructural (proteina NS) . Este tipo de técnica presenta desventajas en cuanto a su costo y optimización, puesto que para su implementación y puesta en marcha se requiere la adquisición de equipamiento y reactivos especializados de elevado costo, como también personal altamente capacitado . Until now, the standard technique for confirming an influenza virus infection corresponds to the molecular analysis of the reverse transcriptase polymerase chain reaction (RT-PCR). For example, the Real-Time RT-PCR Diagnostic Panel for Human Influenza Virus developed by the Center for Disease Control and Prevention (CDC) enables in vitro detection of influenza virus in airway samples from human patients showing signs and symptoms of respiratory infection. This method detects influenza A and B viruses through the reaction of starters against genes encoding highly conserved proteins, such as matrix protein (M protein), nucleoprotein (NP protein), and non-structural protein (NS protein). . This type of technique has disadvantages in terms of cost and optimization, since for its implementation and start-up the acquisition of specialized equipment and high-cost reagents is required, as well as highly trained personnel.
Otro método común para el diagnóstico, es el aislamiento viral en cultivos celulares . El problema de este tipo de técnica es Another common method for diagnosis is viral isolation in cell cultures. The problem with this type of technique is
5 https://espanol.cdc.gov/enes/flu/professionals/diagnosis/rapidlab.htm
que se requiere de equipamiento y personal altamente especializado. Por otro lado, es un método lento que puede entregar resultados diagnósticos dentro de 5 a 14 dias luego de su inicio. 5 https://espanol.cdc.gov/enes/flu/professionals/diagnosis/rapidlab.htm which requires highly specialized equipment and personnel. On the other hand, it is a slow method that can deliver diagnostic results within 5 to 14 days after its start.
En la práctica del diagnóstico clínico, una de las principales dificultades o problemáticas es la muestra misma, puesto que se puede acceder a una cantidad limitada de ella, la que además presenta una baja concentración de antigeno y que incluye la presencia de otras proteínas y componentes celulares que pueden interferir en la reacción de detección. In clinical diagnosis practice, one of the main difficulties or problems is the sample itself, since a limited amount of it can be accessed, which also has a low concentration of antigen and that includes the presence of other proteins and components which may interfere with the detection reaction.
Se han descrito previamente anticuerpos monoclonales para la detección de antigenos del virus de la influenza humana. En el documento W02012045001 (A2 ) , por ejemplo, se divulga un anticuerpo monoclonal humano que se une a la proteina de superficie hemaglutinina . En el documento US9650434B2 se provee de un anticuerpo monoclonal o un fragmento de unión a antigeno del mismo, que se puede unir específicamente al dominio HA1 de la proteina hemaglutinina del subtipo H1 y subtipo H5 de virus de influenza. En ambos documentos, la solución propuesta apunta a la detección de un antigeno diferente de la proteina PB2, y no se demuestra la eficiencia, especificidad y sensibilidad de la unión antigeno-anticuerpo en muestras clínicas . Monoclonal antibodies have been previously described for the detection of antigens of the human influenza virus. In W02012045001 (A2), for example, a human monoclonal antibody is disclosed that binds to the hemagglutinin surface protein. In US9650434B2 a monoclonal antibody or an antigen binding fragment thereof is provided, which can specifically bind to the HA1 domain of the hemagglutinin protein of subtype H1 and subtype H5 of influenza virus. In both documents, the proposed solution aims at the detection of an antigen different from the PB2 protein, and the efficiency, specificity and sensitivity of the antigen-antibody binding in clinical samples are not demonstrated.
En cuanto a la detección de la proteina PB2 de Flu, el documento JP2015189715 (A) proporciona un anticuerpo monoclonal o un fragmento de unión a antigeno del mismo que se une a la subunidad PB2 de la ARN polimerasa dependiente de ARN. Las secuencias de las regiones variables de la cadena pesada y regiones variables de la cadena liviana del anticuerpo allí descrito difieren sustancialmente de las secuencias de los anticuerpos monoclonales parte de la invención. Por otro lado, en JP2015189715 (A) no se realizan ensayos de detección de antigeno
en muestras clínicas de humano, ni se determinan las características de especificidad y sensibilidad de los anticuerpos en el contexto del diagnóstico clínico. Recordemos que en condiciones de diagnóstico clínico se recurre a muestras biológicas que incluyen muy bajas concentraciones de antigeno, lo que dificulta la especificidad y sensibilidad de la reacción antigeno-anticuerpo . As for the detection of Flu's PB2 protein, JP2015189715 (A) provides a monoclonal antibody or an antigen-binding fragment thereof that binds to the PB2 subunit of RNA-dependent RNA polymerase. The sequences of the heavy chain variable regions and light chain variable regions of the antibody described there differ substantially from the sequences of the monoclonal antibodies part of the invention. On the other hand, in JP2015189715 (A) antigen detection tests are not carried out In human clinical samples, the specificity and sensitivity characteristics of the antibodies are not determined in the context of clinical diagnosis. Recall that under clinical diagnostic conditions, biological samples are used that include very low concentrations of antigen, which hinders the specificity and sensitivity of the antigen-antibody reaction.
Se requiere entonces, de una nueva alternativa de diagnóstico del virus de la influenza humana que, a diferencia de las pruebas de diagnóstico molecular y de cultivo celular que conllevan mayores tiempos de respuesta y un elevado costo para su implementación y mantención, permita la detección de una amplia variedad de tipos y subtipos de influenza de forma rápida, sensible, especifica y a un menor costo. Además, aun cuando hasta ahora se han propuesto anticuerpos monoclonales para la detección de otras proteínas de Flu e incluso contra PB2 , estos anticuerpos sólo han sido evaluados en modelos murinos y no corresponden en ningún caso a una solución al problema técnico planteado . Therefore, a new diagnostic alternative for the human influenza virus is required which, unlike molecular diagnostic tests and cell culture tests that entail longer response times and a high cost for their implementation and maintenance, allows the detection of a wide variety of influenza types and subtypes quickly, sensitively, specifically and at a lower cost. Furthermore, even though monoclonal antibodies have been proposed so far for the detection of other Flu proteins and even against PB2, these antibodies have only been evaluated in murine models and do not correspond in any case to a solution to the technical problem posed.
De acuerdo con los antecedentes provistos, se proponen anticuerpos monoclonales que detectan la proteina PB2 para ser utilizados en la detección y diagnóstico rápido, eficaz y certero en pacientes infectados con Flu, donde dichos anticuerpos detectan específicamente la proteina en muestras clínicas a muy bajas concentraciones del antigeno especifico (alta sensibilidad) , incluso distinguiendo el antigeno viral especifico en muestras clínicas que incluyen incluso antigenos de otros virus respiratorios. Adicionalmente, los anticuerpos provistos pueden formar parte de un método de diagnóstico y kit para el diagnóstico de Flu, donde cada uno de los anticuerpos
puede ser utilizado de forma versátil taño como anticuerpo de detección como anticuerpo de captura. According to the background provided, monoclonal antibodies that detect the PB2 protein are proposed to be used in the rapid, effective and accurate detection and diagnosis in patients infected with Flu, where said antibodies specifically detect the protein in clinical samples at very low concentrations of the specific antigen (high sensitivity), even distinguishing the specific viral antigen in clinical samples that even include antigens from other respiratory viruses. Additionally, the provided antibodies can be part of a diagnostic method and kit for the diagnosis of Flu, where each of the antibodies It can be used in a versatile way as a detection antibody as a capture antibody.
Descripción de la invención La presente invención se refiere a anticuerpos monoclonales especificos contra la proteina PB2 o fragmentos de ésta, del virus de influenza humana. De forma particular, la invención corresponde a anticuerpos monoclonales o fragmentos de los mismos secretados por lineas celulares de hibridomas denominados 1A3E2 y 2F11B1, que reconocen la proteina PB2 del virus de la influenza humana (Flu), donde dichos anticuerpos monoclonales o fragmentos de estos comprenden una un anticuerpo que comprende una región variable de cadena ligera donde su CDR1 (CDRLCI) se define según la SEQ ID NO: 1, su CDR2 (CDRLC2) se define por la SEQ ID NO: 2 y su CDR3 (CDRLCS) corresponde a la SEQ ID NO: 3, y una región variable de la cadena pesada donde su CDR1 (CDRHCI) se define según la SEQ ID NO: 4, su CDR2 (CDRHC2) corresponde a laDESCRIPTION OF THE INVENTION The present invention relates to specific monoclonal antibodies against the PB2 protein or fragments thereof, of the human influenza virus. In particular, the invention corresponds to monoclonal antibodies or fragments thereof secreted by hybridoma cell lines called 1A3E2 and 2F11B1, which recognize the PB2 protein of the human influenza virus (Flu), where said monoclonal antibodies or fragments of these comprise an an antibody comprising a light chain variable region where its CDR1 (CDR LCI ) is defined by SEQ ID NO: 1, its CDR2 (CDR LC2 ) is defined by SEQ ID NO: 2 and its CDR3 (CDR LCS ) corresponds to SEQ ID NO: 3, and a variable region of the heavy chain where your CDR1 (CDR HCI ) is defined according to SEQ ID NO: 4, your CDR2 (CDR HC2 ) corresponds to
SEQ ID NO: 5 y su CDR3 (CDRHC3) corresponde a la SEQ ID NO: 6, o un anticuerpo que comprende una región variable de cadena ligera donde su CDR1 (CDRLCI) se define según la SEQ ID NO: 7, su CDR2 ( CDRLC2 ) se define por la SEQ ID NO: 8 y su CDR3 (CDRLCS) corresponde a la SEQ ID NO: 9, y una región variable de la cadena pesada donde su CDR1 (CDRHCI) se define según la SEQ ID NO: 10, su CDR2 (CDRHC2 ) corresponde a la SEQ ID NO: 11 y su CDR3 (CDRHCS) corresponde a la SEQ ID NO: 12, donde el anticuerpo puede ser usado como anticuerpo de detección o anticuerpo de captura. Adicionalmente, se proporciona un método de diagnóstico de infección por Flu en una muestra biológica que utiliza los anticuerpos monoclonales en formato de kit de diagnóstico para detectar Flu, donde dicho kit comprende al menos un anticuerpo monoclonal contra Flu de acuerdo a lo descrito previamente.
Los anticuerpos descritos en la invención presentan importantes características técnicas ventajosas y destacables con respecto a otros anticuerpos y métodos de detección de antigenos virales ya existentes. SEQ ID NO: 5 and its CDR3 (CDR HC3 ) corresponds to SEQ ID NO: 6, or an antibody that comprises a light chain variable region where its CDR1 (CDR LCI ) is defined according to SEQ ID NO: 7, its CDR2 (CDR LC2) is defined by SEQ ID NO: 8 and its CDR3 (CDR LCS ) corresponds to SEQ ID NO: 9, and a variable region of the heavy chain where its CDR1 (CDR HCI ) is defined according to SEQ ID NO: 10, its CDR2 (CDR HC2) corresponds to SEQ ID NO: 11 and its CDR3 (CDR HCS ) corresponds to SEQ ID NO: 12, where the antibody can be used as detection antibody or capture antibody. Additionally, a method of diagnosing Flu infection in a biological sample is provided that uses the monoclonal antibodies in diagnostic kit format to detect Flu, where said kit comprises at least one monoclonal antibody against Flu as previously described. The antibodies described in the invention present important advantageous and remarkable technical characteristics with respect to other antibodies and detection methods of viral antigens that already exist.
En primer lugar, cada virus tiene proteínas de superficie especificas, por tanto, otras técnicas de diagnóstico basadas en anticuerpos monoclonales para otros tipos de virus respiratorios no son comparables a la invención propuesta. La especificidad de detección de los anticuerpos contra la proteina PB2 de Flu respecto de otros antigenos virales, por ejemplo contra la proteina pIII de adenovirus, se demuestra en los resultados provistos en las figuras 1A, IB y 1C . A partir de estos resultados es posible concluir que los anticuerpos provistos en el alcance de la presente solicitud sólo reconocen a la proteina PB2 de Flu, y que en ensayos de ELISA con antigenos del virus ADV no se observó señal de detección. First, each virus has specific surface proteins, therefore, other diagnostic techniques based on monoclonal antibodies for other types of respiratory viruses are not comparable to the proposed invention. The detection specificity of the antibodies against the Flu PB2 protein relative to other viral antigens, for example against the adenovirus pIII protein, is demonstrated in the results provided in Figures 1A, IB and 1C. From these results it is possible to conclude that the antibodies provided in the scope of the present application only recognize Flu's PB2 protein, and that in ELISA tests with ADV virus antigens no detection signal was observed.
En segundo lugar, los anticuerpos parte del alcance de la invención permiten detectar de forma especifica a la proteina PB2 o fragmentos de ésta, de tal forma que no compiten entre si por el sitio de unión al antigeno, ni ejercen un impedimento para unirse simultáneamente a este. Second, the antibodies that are part of the scope of the invention make it possible to specifically detect the PB2 protein or fragments thereof, so that they do not compete with each other for the antigen binding site, nor do they impede their simultaneous binding to East.
En tercer lugar, permiten detectar la proteina PB2 o fragmentos de ésta con alta sensibilidad en muestras que contengan escasa cantidad de antigeno, como muestras de hisopado nasofaríngeo por ejemplo . Third, they allow the detection of the PB2 protein or fragments of it with high sensitivity in samples that contain a low amount of antigen, such as nasopharyngeal swab samples, for example.
Los anticuerpos monoclonales propuestos son capaces de detectar a la proteina PB2 , una proteina altamente conservada. La estrategia de detección de una proteina viral conservada permite que los anticuerpos parte del alcance de la invención detecten diferentes tipos de influenza humana, entre ellos, influenza A, B y C.
Cuando en la presente invención se hace referencia a secuencias CDR estas corresponden a secuencias cortas que se encuentran en los dominios variables de las proteínas que presentan función de detección de antigenos . Se presentan las secuencias CDR para la cadena pesada (CDRHC) y la cadena liviana (CDRi,c)de los anticuerpos secretados por los hibridomas 1A3E2 y 2F11B1. The proposed monoclonal antibodies are capable of detecting PB2 protein, a highly conserved protein. The detection strategy of a conserved viral protein allows antibodies that are within the scope of the invention to detect different types of human influenza, including influenza A, B, and C. When CDR sequences are referred to in the present invention, they correspond to short sequences that are found in the variable domains of proteins that have antigen detection function. The CDR sequences for heavy chain (CDR HC ) and light chain (CDRi, c) of the antibodies secreted by 1A3E2 and 2F11B1 hybridomas are presented.
Los anticuerpos monoclonales descritos pueden ser utilizados para ensayos de detección, diagnóstico y/o determinación de infección por Flu. Estos anticuerpos pueden ser utilizados simultáneamente para incrementar la sensibilidad de detección en muestras clínicas donde existe escasa cantidad y disponibilidad de antigeno. Al respecto, se proporciona también un método de diagnóstico de infección de Flu en una muestra biológica, el cual comprende poner en contacto la muestra biológica con el anticuerpo monoclonal contra la proteina PB2 de Flu o un fragmento de él de acuerdo a la reivindicación, y detectar la unión del anticuerpo con el antigeno. La muestra biológica puede corresponder, sin limitarse, a células ín vítro infectadas con Flu, secreciones nasales, lavados nasales, liquido cefalorraquídeo, secreciones faríngeas y/o lavados o secreciones bronquiales. Como parte del método, el ensayo utilizado para la detección de la unión antigeno-anticuerpo se selecciona de ELISA, Luminex, inmunofluorescencia, inmunohistoquimica, inmunocromatografia, citometria de flujo, cell sorter, inmunoprecipitación y/o Western blot . The described monoclonal antibodies can be used for detection, diagnosis and / or determination of Flu infection. These antibodies can be used simultaneously to increase detection sensitivity in clinical samples where there is little quantity and availability of antigen. In this regard, there is also provided a method of diagnosing Flu infection in a biological sample, which comprises contacting the biological sample with the monoclonal antibody against Flu PB2 protein or a fragment thereof according to claim, and detect binding of the antibody to the antigen. The biological sample may correspond, without limitation, to in vitro cells infected with Flu, nasal secretions, nasal lavages, cerebrospinal fluid, pharyngeal secretions and / or bronchial lavages or secretions. As part of the method, the assay used for detection of antigen-antibody binding is selected from ELISA, Luminex, immunofluorescence, immunohistochemistry, immunochromatography, flow cytometry, cell sorter, immunoprecipitation, and / or Western blot analysis.
La presente invención incluye también un kit de diagnóstico para detectar al virus de la influenza humana, el que comprende: un anticuerpo monoclonal contra la proteina PB2 de Flu o un fragmento de la misma, donde dicho anticuerpo puede actuar como anticuerpo de captura o de detección, donde particularmente, el anticuerpo de detección se encuentra conjugado a un marcador
para su detección; un soporte sólido al cual se adosa el anticuerpo; y reactivos para detectar el marcador incluido en el anticuerpo de detección, tales como fluoróforos, biotina, radioisótopos, metales y enzimas. The present invention also includes a diagnostic kit for detecting human influenza virus, which comprises: a monoclonal antibody against Flu PB2 protein or a fragment thereof, where said antibody can act as a capture or detection antibody , where particularly, the detection antibody is conjugated to a marker for detection; a solid support to which the antibody is attached; and reagents for detecting the marker included in the detection antibody, such as fluorophores, biotin, radioisotopes, metals, and enzymes.
En la presente invención cuando se refiere a anticuerpo de captura, éste corresponde al anticuerpo que se une especif eamente al antigeno. En el caso del anticuerpo de detección este corresponde al anticuerpo al cual se le conjuga un marcador para ser detectado por diferentes test tales como test inmunocromatográfico, luminex, citometria de flujo, inmunofluorescencia, radioinmunoanálisis , Western blot, Dot plot, ELISA, luminex, inmunodifusión o inmunoprecipitación . In the present invention when referring to capture antibody, this corresponds to the antibody that specifically binds to the antigen. In the case of the detection antibody, this corresponds to the antibody to which a marker is conjugated to be detected by different tests such as immunochromatographic test, luminex, flow cytometry, immunofluorescence, radioimmunoassay, Western blot, Dot plot, ELISA, luminex, immunodiffusion. or immunoprecipitation.
Los anticuerpos parte de la presente invención pueden actuar de forma dual como anticuerpo de captura o como anticuerpo de detección cuando se acopla a marcador de detección. El marcador de detección estará conjugado al anticuerpo de detección, y éste puede corresponder, sin limitarse, a fluoróforos, biotina, radioisótopos, metales y enzimas. De forma preferente, el anticuerpo de detección se conjuga al sistema reportero basado en la detección de la actividad de la enzima peroxidasa de rábano picante (HRP, por sus siglas en inglés) . The antibodies part of the present invention can dual function as a capture antibody or as a detection antibody when coupled to the detection marker. The detection marker will be conjugated to the detection antibody, and this can correspond, without limitation, to fluorophores, biotin, radioisotopes, metals and enzymes. Preferably, the detection antibody is conjugated to the reporter system based on detection of activity of horseradish peroxidase enzyme (HRP).
Descripción de las figuras de la invención Description of the figures of the invention
Figura 1 : Detección de proteina PB2 de Flu por los anticuerpos monoclonales producidos por los hibridomas 1A3E2 y 2F11B1, mediante un ensayo de ELISA indirecto. La placa fue activada con 50 ng de proteina PB2 de Flu recombinante purificada, 50 ng de proteina pIII de ADV (como control de especificidad) y 20 pg de células MDCK no infectadas (utilizadas como control de especificidad) e infectadas con Flu. Se incluyeron pocilios control sin antigeno, con anticuerpo primario, con anti-IgG de
ratón conjugado con HRP (sin activar) y pocilios sin antigeno ni anticuerpo primario, solo con anticuerpo anti-IgG de ratón (HRP), datos no mostrados en el gráfico. Posteriormente, los pocilios se incubaron con los anticuerpos anti-PB2 provenientes del hibridoma 1A3E2, en cantidad de 170 ng (A), el hibridoma 2F11B1 en cantidad de 170 ng (B) y el anticuerpo policlonal comercial Anti-Influenza A virus PB2 protein antibody, número de catálogo GTX125926 (GeneTex) utilizado en cantidad de 170 ng (C) . Los datos mostrados en el gráfico expresan la absorbancia (en OD, densidad óptica) detectada a 450 nm, emitida por la conversión del sustrato Tetrametilbenzidina a un compuesto coloreado, catalizada por la enzima Horseradish peroxidase (HRP) conjugada en un anticuerpo secundario anti-IgG de ratón que se unió específicamente a los anticuerpos secretados por los hibridomas 1A3E2, 4D8C6 y GTX125926 de GeneTex. Los valores corresponden al promedio +/- la desviación estándar de la absorbancia emitida por cada muestra en al menos dos experimentos independientes. Donde, * P<0.05; ** P<0,01; *** P<0, 001 y **** P <0,0001 por el test de student paramétrico comparado los resultados de la proteina pIII-ADV versus los de PB2-FÍU, y por otro lado comparando las células no infectadas versus infectadas . Figure 1: Detection of Flu PB2 protein by monoclonal antibodies produced by hybridomas 1A3E2 and 2F11B1, by means of an indirect ELISA assay. The plate was activated with 50 ng of purified recombinant Flu PB2 protein, 50 ng of ADV pIII protein (as a specificity control) and 20 pg of uninfected MDCK cells (used as a specificity control) and infected with Flu. Control wells without antigen, with primary antibody, with anti-IgG were included. mouse conjugated to HRP (not activated) and wells without antigen or primary antibody, only with anti-mouse IgG antibody (HRP), data not shown in the graph. Subsequently, the wells were incubated with the anti-PB2 antibodies from the 1A3E2 hybridoma, in the amount of 170 ng (A), the 2F11B1 hybridoma in the amount of 170 ng (B) and the commercial polyclonal antibody Anti-Influenza A virus PB2 protein antibody , catalog number GTX125926 (GeneTex) used in quantity of 170 ng (C). The data shown in the graph expresses the absorbance (in OD, optical density) detected at 450 nm, emitted by the conversion of the substrate Tetramethylbenzidine to a colored compound, catalyzed by the enzyme Horseradish peroxidase (HRP) conjugated in a secondary anti-IgG antibody mouse that specifically bound to the antibodies secreted by GeneTex hybridomas 1A3E2, 4D8C6 and GTX125926. Values correspond to the average +/- standard deviation of absorbance emitted by each sample in at least two independent experiments. Where, * P <0.05; ** P <0.01; *** P <0, 001 and **** P <0.0001 by the parametric student test comparing the results of the pIII-ADV protein versus those of PB2-FÍU, and on the other hand comparing the uninfected cells versus infected.
Figura 2 : Determinación de sensibilidad de anticuerpos monoclonales producidos por los hibridomas 1A3E2 y 2F11B1 en la detección de la proteina PB2 de Flu. Placas de ELISA se activaron con diluciones seriadas 1:2, iniciando con 50 ng de proteina PB2 y finalizando con 0,04 ng. Posteriormente, los pocilios se incubaron con los anticuerpos anti-PB2 provenientes del hibridoma 1A3E2, en cantidad de 170 ng (A) y el hibridoma 2F11B1 en cantidad de 170 ng (B) . Se incluyeron pocilios sin activar como control negativo. Los datos mostrados en el gráfico expresan la absorbancia a 450 nm emitida por la conversión del sustrato
Tetrametilbenzidina a un compuesto coloreado catalizada por la enzima Horseradish peroxidase (HRP) conjugada a los anticuerpos anti-PB2 provenientes de los hibridomas 1A3E2 y 2F11B1 en cantidad de 170 ng (A y B) . Los valores corresponden al promedio +/- la desviación estándar de la absorbancia emitida por cada muestra en al menos dos experimentos independientes. * P<0,05; ** P<0, 01; *** P<0,001 por el test de student paramétrico comparando los resultados del pocilio denominado control versus cada una de las diluciones de la proteina PB2. Figure 2: Determination of sensitivity of monoclonal antibodies produced by 1A3E2 and 2F11B1 hybridomas in the detection of Flu PB2 protein. ELISA plates were activated with 1: 2 serial dilutions, starting with 50 ng of PB2 protein and ending with 0.04 ng. Subsequently, the wells were incubated with the anti-PB2 antibodies from the 1A3E2 hybridoma, in the amount of 170 ng (A) and the 2F11B1 hybridoma in the amount of 170 ng (B). Unactivated wells were included as a negative control. The data shown in the graph express the absorbance at 450 nm emitted by the conversion of the substrate Tetramethylbenzidine to a colored compound catalyzed by the Horseradish peroxidase (HRP) enzyme conjugated to anti-PB2 antibodies from hybridomas 1A3E2 and 2F11B1 in an amount of 170 ng (A and B). Values correspond to the average +/- standard deviation of absorbance emitted by each sample in at least two independent experiments. * P <0.05; ** P <0.01; *** P <0.001 by the parametric student test comparing the results of the well called control versus each of the dilutions of the PB2 protein.
Figura 3: Ensayo de diluciones seriadas de los anticuerpos monoclonales anti-PB2 de Flu producidos por los hibridomas 1A3E2 y 2F11B1, para la detección de antigenos purificados de Flu. Se activaron placas de ELISA con 50 ng de proteina recombinante PB2 de Flu y se detectó el antigeno con 11 diluciones seriadas 1:2 de los anticuerpos anti-PB2 1A3E2 (A) ó 2F11B1 (B) , partiendo desde una concentración de 3,4 mr/hIE (170 ng por pocilio) . Los datos se expresan como el promedio +/- la desviación estándar del valor de la absorbancia emitida a 450 nm de cada muestra en duplicado, en al menos dos experimentos independientes. * P<0,05; ** P<0,01 y *** P<0,001 por el test de student paramétrico comparando los resultados del pocilio denominado control versus cada una de las diluciones de la proteina PB2. Figure 3: Assay of serial dilutions of Flu anti-PB2 monoclonal antibodies produced by 1A3E2 and 2F11B1 hybridomas, for the detection of purified Flu antigens. ELISA plates were activated with 50 ng of recombinant Flu PB2 protein and the antigen was detected with 11 serial dilutions 1: 2 of the anti-PB2 antibodies 1A3E2 (A) or 2F11B1 (B), starting from a concentration of 3.4 mr / hIE (170 ng per well). Data are expressed as the average +/- standard deviation of the absorbance value emitted at 450 nm for each sample in duplicate, in at least two independent experiments. * P <0.05; ** P <0.01 and *** P <0.001 by the parametric student test comparing the results of the well called control versus each of the dilutions of the PB2 protein.
Figura 4 : Detección de Flu en muestras clínicas mediante ELISA en Sándwich, utilizando la combinación de los anticuerpos monoclonales secretados por los hibridomas 1A3E2 y 2F11B1.Figure 4: Detection of Flu in clinical samples by sandwich ELISA, using the combination of monoclonal antibodies secreted by hybridomas 1A3E2 and 2F11B1.
Placas de ELISA fueron activadas con 170 ng de anticuerpo secretado por el hibridoma 1A3E2 (anti-Flu) , funcionando como anticuerpo de captura. Los pocilios activados con el anticuerpo de captura se incubaron con 50 mΐ de muestras de hisopado nasofaríngeo (HNF) de pacientes que presentaban cuadros respiratorios virales. Como controles negativos se analizaron 10
muestras de controles sanos. Se utilizaron 12 muestras de pacientes positivos para Flu y como control de especificidad, se incluyeron 3 muestras de pacientes positivos para el virus Parainfluenza. Como control positivo se incluyeron pocilios a los que se añadió proteina recombinante PB2-Flu purificada. Para la detección de la proteina capturada por el anticuerpo 1A3E2, se utilizaron los anticuerpos producidos por el hibridoma 2F11B1, conjugados a la enzima Horseradish Peroxidase, en una dilución 1:2000 (1,8 ng/mΐ por pocilio). Los datos mostrados es la mediana del valor de la absorbancia emitida a 450 nm de cada muestra (**P<0,01 **** y p <0,0001; mediante el test de student no paramétrico y post test de Mann Whitney comparando pacientes positivos de Flu versus controles sanos, y frente a los virus usados como control de especificidad) . ELISA plates were activated with 170 ng of antibody secreted by the 1A3E2 hybridoma (anti-Flu), functioning as a capture antibody. The wells activated with the capture antibody were incubated with 50 mΐ of nasopharyngeal swab (HNF) samples from patients presenting with viral respiratory symptoms. As negative controls, 10 samples from healthy controls. Twelve samples from Flu-positive patients were used and as a specificity control, 3 samples from patients positive for Parainfluenza virus were included. As a positive control, wells were included to which purified recombinant PB2-Flu protein was added. For the detection of the protein captured by the 1A3E2 antibody, the antibodies produced by the 2F11B1 hybridoma, conjugated to the Horseradish Peroxidase enzyme, were used in a 1: 2000 dilution (1.8 ng / mΐ per well). The data shown is the median value of the absorbance emitted at 450 nm for each sample (** P <0.01 **** and p <0.0001; using the non-parametric student test and the Mann Whitney post test comparing Flu positive patients versus healthy controls, and against the viruses used as specificity control).
Figura 5: Detección de proteina PB2 mediante ensayo de ELISA indirecto, utilizando los anticuerpos monoclonales completos y fragmentos de ellos, secretados por los hibridomas 1A3E2 y 2F11B1 conjugados con biotina. Se observa la detección de la proteina PB2 de los anticuerpos conjugados con Biotina. En negro y blanco se indican fragmentos del anticuerpo secretados por los hibridomas 1A3E2 y 2F11B1 respectivamente. Mientras que en gris se presenta la actividad de los fragmentos completos de los anticuerpos secretados por los hibridomas 1A3E2 y 2F11B1 respectivamante . Los datos mostrados en el gráfico expresan la absorbancia a 450 nm emitida por la conversión del sustrato Tetrametilbenzidina a un compuesto coloreado catalizada por la enzima Horseradish peroxidase (HRP) . Se muestra el promedio del valor de la absorbancia emitida a 450 nm de cada muestra (donde b es igual a p<0,0001 comparado con a; mediante la prueba de ANOVA de 2 vias comparando el pocilio sin muestra versus el pocilio con proteina con todos los anticuerpos).
Ejemplos que permiten demostrar las distintas aplicaciones de los anticuerpos monoclonales de la invención. Figure 5: Detection of PB2 protein by indirect ELISA assay, using whole monoclonal antibodies and fragments of them, secreted by biotin-conjugated 1A3E2 and 2F11B1 hybridomas. Detection of PB2 protein from Biotin-conjugated antibodies is observed. Fragments of the antibody secreted by hybridomas 1A3E2 and 2F11B1 respectively are indicated in black and white. While in gray the activity of the complete fragments of the antibodies secreted by the 1A3E2 and 2F11B1 hybridomas respectively is presented. The data shown in the graph expresses the absorbance at 450 nm emitted by the conversion of the substrate Tetramethylbenzidine to a colored compound catalyzed by the enzyme Horseradish peroxidase (HRP). The average of the absorbance value emitted at 450 nm of each sample is shown (where b is equal to p <0.0001 compared to a; by means of the 2-way ANOVA test comparing the well without sample versus the well with protein with all antibodies). Examples that demonstrate the different applications of the monoclonal antibodies of the invention.
Ejemplo 1: Determinación de la secuencia nucleotidica que codifica las cadenas livianas (VL) y pesadas (VH) de la región variable del anticuerpo anti PB2 Flu secretado por el hibridoma 1A3E2. Example 1: Determination of the nucleotide sequence encoding the light (VL) and heavy (VH) chains of the variable region of the anti PB2 Flu antibody secreted by the 1A3E2 hybridoma.
El hibridoma 1A3E2 se creció en medio de cultivo DMEM-high glucose suplementado con 3,7 g/L de Bicarbonato de Sodio y 10% de suero fetal bovino, a 37° C con 10% CO2, hasta una densidad celular de 700.000 células/mL. Se obtuvo el RNA total de 3,5 xlO6 células, realizando un tratamiento con el compuesto Trizol (Invitrogen) . Se utilizó 0,5 mg de RNA para generar el cDNA mediante reacción de retrotranscripción con el kit PrimeScriptTM lst Strand cDNA Synthesis, el cual utiliza partidores universales específicos del isotipo. La cadena liviana y pesada del anticuerpo se amplificaron de acuerdo con el procedimiento operativo estándar (SOP) de amplificación rápida de los extremos de cDNA (RACE) de GenScript. Los fragmentos de anticuerpo amplificados se clonaron por separado en un vector de clonación estándar. Se realizó una PCR de colonias para identificar clones que tuvieran insertos con el tamaño correcto. Al menos cinco colonias con insertos de tamaños correctos se secuenciaron por cada fragmento. Se alinearon las secuencias de diferentes clones y se proporcionó la secuencia de consenso de estos clones . Las secuencias nucleotidicas de las cadenas pesada y liviana de los anticuerpos secretados por el hibridoma 1A3E2 fueron identificadas, siendo identificadas con las SEQ ID NO. 1 y SEQ
ID NO.3 para el caso de las cadenas pesadas y SEQ ID NO. 2 y SEQ ID NO.4 para el caso de las cadenas livianas. The 1A3E2 hybridoma was grown in DMEM-high glucose culture medium supplemented with 3.7 g / L Sodium Bicarbonate and 10% fetal bovine serum, at 37 ° C with 10% CO2, up to a cell density of 700,000 cells / mL. The total RNA of 3.5 x 10 cells was obtained, carrying out a treatment with the compound Trizol (Invitrogen). 0.5 mg of RNA was used to generate the cDNA by reverse transcription reaction with the PrimeScriptTM lst Strand cDNA Synthesis Kit, which uses isotype specific universal cleavers. The light and heavy chain of the antibody were amplified according to the GenScript cDNA Extreme Rapid Amplification (RACE) Standard Operating Procedure (SOP). Amplified antibody fragments were cloned separately into a standard cloning vector. Colony PCR was performed to identify clones that had inserts of the correct size. At least five colonies with correct size inserts were sequenced for each fragment. The sequences of different clones were aligned and the consensus sequence of these clones was provided. The nucleotide sequences of the heavy and light chains of the antibodies secreted by the 1A3E2 hybridoma were identified, being identified with SEQ ID NO. 1 and SEQ ID NO.3 for the case of heavy chains and SEQ ID NO. 2 and SEQ ID NO.4 in the case of light chains.
Ejemplo 2 : Determinación de la secuencia nucleotidica que codifica las cadenas livianas (VL) y pesadas (VH) de la región variable del anticuerpo anti PB2 Flu secretado por el hibridoma 2F11B1. Example 2: Determination of the nucleotide sequence encoding the light (VL) and heavy (VH) chains of the variable region of the anti-PB2 Flu antibody secreted by the 2F11B1 hybridoma.
El hibridoma 2F11B1 se creció en medio de cultivo DMEM-high glucose suplementado con 3,7 g/L de Bicarbonato de Sodio y 10% de suero fetal bovino, a 37° C con 10% CO2, hasta una densidad celular de 700.000 células/mL. Se obtuvo el RNA total de 3,5 xlO6 células, realizando un tratamiento con el compuesto Trizol (Invitrogen) . Se utilizó 0,5 mg de RNA para generar el cDNA mediante reacción de retrotranscripción con el kit PrimeScriptTM lst Strand cDNA Synthesis, el cual utiliza partidores universales específicos del isotipo. La cadena liviana y pesada del anticuerpo se amplificaron de acuerdo con el procedimiento operativo estándar (SOP) de amplificación rápida de los extremos de cDNA (RACE) de GenScript. Los fragmentos de anticuerpo amplificados se clonaron por separado en un vector de clonación estándar. Se realizó una PCR de colonias para identificar clones que tuvieran insertos con el tamaño correcto. Al menos cinco colonias con insertos de tamaños correctos se secuenciaron por cada fragmento. Se alinearon las secuencias de diferentes clones y se proporcionó la secuencia de consenso de estos clones . A partir de esto, se determinaron las secuencias nucleotidicas de las cadenas pesada y liviana de los anticuerpos secretados por el hibridoma 2F11B1, correspondiendo las secuencias identificadas como SEQ ID NO. 1 y SEQ ID NO .3 a las cadenas livianas y las secuencias identificadas como SEQ ID NO. 1 y SEQ ID NO.3 a las cadenas pesadas.
Ejemplo 3: Ensayo de detección de antigenos de Flu, determinación de especificidad de los anticuerpos monoclonales anti-PB2 de Flu para antigenos purificados de Flu mediante ensayo de ELISA indirecto. The 2F11B1 hybridoma was grown in DMEM-high glucose culture medium supplemented with 3.7 g / L Sodium Bicarbonate and 10% fetal bovine serum, at 37 ° C with 10% CO2, up to a cell density of 700,000 cells / mL. The total RNA of 3.5 x 10 cells was obtained, carrying out a treatment with the compound Trizol (Invitrogen). 0.5 mg of RNA was used to generate the cDNA by reverse transcription reaction with the PrimeScriptTM lst Strand cDNA Synthesis Kit, which uses isotype specific universal cleavers. The light and heavy chain of the antibody were amplified according to the GenScript cDNA Extreme Rapid Amplification (RACE) Standard Operating Procedure (SOP). Amplified antibody fragments were cloned separately into a standard cloning vector. Colony PCR was performed to identify clones that had inserts of the correct size. At least five colonies with correct size inserts were sequenced for each fragment. The sequences of different clones were aligned and the consensus sequence of these clones was provided. From this, the nucleotide sequences of the heavy and light chains of the antibodies secreted by the 2F11B1 hybridoma were determined, corresponding to the sequences identified as SEQ ID NO. 1 and SEQ ID NO .3 to the light chains and the sequences identified as SEQ ID NO. 1 and SEQ ID NO.3 to heavy chains. Example 3: Flu antigen detection assay, determination of specificity of Flu anti-PB2 monoclonal antibodies for purified Flu antigens by indirect ELISA assay.
Este ensayo tiene como objetivo demostrar la especificidad por la proteina PB2 de Flu de los anticuerpos producidos por los hibridomas 1A3E2 y 2F11B1. La detección del antigeno se llevó a cabo mediante la técnica de ELISA indirecto, donde la placa de ELISA se activó con 50 ng de antigeno purificado por 1 hora a 37°C. De igual manera se activó la placa con 20 pg de Usado celular de células MDCK no infectadas (como control negativo) e infectadas con virus Flu serotipo A. Otro control negativo incluido fue 50 ng de proteina pIII de ADV en un pocilio independiente. Posteriormente, la placa se lavó dos veces con buffer fosfato salino (PBS) lX/Tween20 0,05%. Luego la placa se bloqueó por 2 horas a 37°C con PBS IX/ Suero Fetal Bovino (FBS) 10%. Posteriormente se repitieron los lavados y a continuación se incubaron cada uno de los anticuerpos (1A3E2 y 2F11B1) a una concentración final de 3,4 pg/mL (170 ng por pocilio), diluidos en PBS 1X/FBS 10%, por 1 hora a 37°C (cada anticuerpo en una placa independiente). Bajo las mismas condiciones, en una placa diferente, se realizó un ensayo control utilizando un anticuerpo monoclonal comercial que reconoce la proteina PB2 de Flu (Anti influenza A virus PB2 protein antibody, número de catálogo GTX125926, GeneTex) a una concentración de 3,4 pg/mL. Transcurrido el tiempo de incubación, se repitieron los lavados y se agregó a cada uno de los pocilios un anticuerpo secundario anti-IgG de ratón marcado con la enzima peroxidasa de rábano (Horseradish peroxidase, HRP) en dilución 1 en 2000 (1,8 ng/mΐ por pocilio) en PBS 1X/FBS 10%, por 1 hora a temperatura ambiente
( 25°C) , en oscuridad. Finalmente, se realizaron los lavados y se reveló con 50 pL de buffer citrato/tetrametil-benzidina (TMB, 3-3' -5-5' tetramethylbenzidine, lmg/mL, Becton Dickinson) . Para detener la reacción se adicionaron 50 pL de H2SO4 2N y el resultado se leyó en un lector de ELISA, a 450nm. Para determinar que la reacción del anticuerpo secundario era especifica en reconocer al anticuerpo primario y además que la señal obtenida no sea provocada por unión inespecifica del anticuerpo secundario al antigeno viral, se realizaron controles en los cuales se utilizó solamente el anticuerpo secundario sin anticuerpo primario ni muestra (pocilio sin activar) . Otro control para determinar que la reacción del anticuerpo primario es especifica para el antigeno, consistió en el uso de los anticuerpos sobre una placa de ELISA que no ha sido activada con el antigeno (sin antigeno) o usando los anticuerpos sobre una placa de ELISA que poseía 50 ng de la proteina pIII de ADV ó células no infectadas . Los resultados muestran que los anticuerpos monoclonales de la invención son capaces de reconocer 50 ng de antigeno purificado, de manera especifica, ya que no reconocen la proteina pIII de ADV, ni proteínas de células no infectadas (Figura 1A y IB) . Por otro lado se observó que el anticuerpo comercial (Figura 1C) utilizado en el ensayo como control, aunque fue especifico para la detección sólo de las células infectadas, no fue eficiente en detectar la proteina PB2 de Flu recombinante purificada en nuestro laboratorio. Todos los controles negativos utilizados entregaron resultados esperados (datos no mostrados en las figuras) . This assay aims to demonstrate the specificity for Flu PB2 protein of the antibodies produced by hybridomas 1A3E2 and 2F11B1. Antigen detection was carried out using the indirect ELISA technique, where the ELISA plate was activated with 50 ng of purified antigen for 1 hour at 37 ° C. Likewise, the plate was activated with 20 pg of Cell used from uninfected MDCK cells (as a negative control) and infected with Flu serotype A virus. Another negative control included was 50 ng of ADV pIII protein in a separate well. Subsequently, the plate was washed twice with phosphate buffered saline (PBS) lX / Tween20 0.05%. The plate was then blocked for 2 hours at 37 ° C with PBS IX / 10% Fetal Bovine Serum (FBS). Subsequently, the washes were repeated and each of the antibodies (1A3E2 and 2F11B1) were then incubated at a final concentration of 3.4 pg / mL (170 ng per well), diluted in PBS 1X / FBS 10%, for 1 hour at 37 ° C (each antibody on a separate plate). Under the same conditions, on a different plate, a control assay was performed using a commercial monoclonal antibody that recognizes Flu's PB2 protein (catalog number GTX125926, GeneTex) at a concentration of 3.4. pg / mL. At the end of the incubation time, the washings were repeated and a secondary horseradish peroxidase enzyme-labeled horseradish peroxidase (HRP) antibody in dilution 1 in 2000 (1.8 ng) was added to each well. / mΐ per well) in PBS 1X / FBS 10%, for 1 hour at room temperature (25 ° C), in darkness. Finally, the washes were performed and revealed with 50 pL of citrate / tetramethyl-benzidine buffer (TMB, 3-3 '-5-5' tetramethylbenzidine, lmg / mL, Becton Dickinson). To stop the reaction, 50 pL of H2SO4 2N were added and the result was read in an ELISA reader, at 450nm. To determine that the reaction of the secondary antibody was specific to recognize the primary antibody and also that the signal obtained was not caused by nonspecific binding of the secondary antibody to the viral antigen, controls were performed in which only the secondary antibody was used without primary antibody nor sample (well not activated). Another check to determine that the reaction of the primary antibody is specific for the antigen, consisted of using the antibodies on an ELISA plate that has not been activated with the antigen (without antigen) or using the antibodies on an ELISA plate that he had 50 ng of the pIII protein from ADV or uninfected cells. The results show that the monoclonal antibodies of the invention are able to recognize 50 ng of purified antigen, specifically, since they do not recognize the ADV pIII protein, nor proteins from uninfected cells (Figure 1A and IB). On the other hand, it was observed that the commercial antibody (Figure 1C) used in the assay as a control, although it was specific for the detection only of infected cells, was not efficient in detecting the purified recombinant Flu PB2 protein in our laboratory. All the negative controls used gave expected results (data not shown in the figures).
Ejemplo 4 : Ensayo para determinar la sensibilidad de los anticuerpos monoclonales para la detección de antigenos virales anti-PB2 de Flu.
El ensayo se realizó para determinar la máxima dilución de proteina que los anticuerpos monoclonales anti-PB2 de Flu provenientes de los hibridomas 1A3E2 y 2F11B1 son capaces de detectar mediante ELISA indirecto. Para esto, se ocupó la misma técnica descrita en el ejemplo 3. Se activó la placa con 11 diluciones seriadas 1:2 de proteina PB2 de Flu, partiendo con 50 ng de antigeno purificado. Los anticuerpos anti-PB2 1A3E2 ó 2F11B1, se utilizaron en una concentración de 3,4 pg/mL(170 ng/pocillo), diluidos en PBS 1X/FBS 10%. Posteriormente se adicionó el anticuerpo de detección anti-IgG de ratón en una dilución de 1:2.000 (1,8 ng/mΐ por pocilio) y se incubo 1 hora a temperatura ambiente ( 25°C) , en oscuridad. Finalmente, se realizaron los lavados y se reveló con 50 pL de buffer citrato/Tetrametilbenzidina (TMB, 3-3' -5-5' tetrametilbenzidina, lmg/mL, Becton Dickinson) . Para detener la reacción se adicionaron 50 pL de H2SO4 2N y el resultado se leyó en un lector de ELISA, a 450nm. Los resultados mostraron que el anticuerpo anti-PB2 1A3E2 es capaz de reconocer hasta 780 picogramos (pg) de la proteina PB2 de Flu (Figura 2A) . El anticuerpo anti-PB2 proveniente del hibridoma 2F11B1, mostró la misma sensibilidad que el anticuerpo anti-PB2 1A3E2 (Figura 2B) . Se incluyeron en todos los ensayos controles que permitieran descartar reacciones inespecificas tanto de los anticuerpos, los cuales contenían todos componentes del ensayo exceptuando la muestra (proteina PB2 Flu, datos no mostrados en los gráficos) . Example 4: Assay to determine the sensitivity of monoclonal antibodies for the detection of Flu anti-PB2 viral antigens. The assay was performed to determine the maximum protein dilution that Flu anti-PB2 monoclonal antibodies from 1A3E2 and 2F11B1 hybridomas are capable of detecting by indirect ELISA. For this, the same technique described in Example 3 was used. The plate was activated with 11 serial dilutions 1: 2 of Flu PB2 protein, starting with 50 ng of purified antigen. Anti-PB2 1A3E2 or 2F11B1 antibodies were used at a concentration of 3.4 pg / mL (170 ng / well), diluted in 1X PBS / 10% FBS. Subsequently, the anti-mouse IgG detection antibody was added at a dilution of 1: 2,000 (1.8 ng / mΐ per well) and incubated 1 hour at room temperature (25 ° C), in the dark. Finally, washes were performed and revealed with 50 pL of citrate / Tetramethylbenzidine buffer (TMB, 3-3 '-5-5' tetramethylbenzidine, lmg / mL, Becton Dickinson). To stop the reaction, 50 pL of H2SO4 2N were added and the result was read in an ELISA reader, at 450nm. The results showed that the anti-PB2 antibody 1A3E2 is capable of recognizing up to 780 picograms (pg) of the Flu2 PB2 protein (Figure 2A). The anti-PB2 antibody from the 2F11B1 hybridoma showed the same sensitivity as the anti-PB2 1A3E2 antibody (Figure 2B). Controls that allowed to rule out nonspecific reactions of both the antibodies, which contained all components of the assay except the sample (PB2 Flu protein, data not shown in the graphs) were included in all the tests.
Ejemplo 5: Ensayo para determinar la eficiencia de los anticuerpos monoclonales para detectar antigenos virales de Flu, mediante ELISA indirecto. Example 5: Assay to determine the efficiency of monoclonal antibodies to detect Flu viral antigens, by indirect ELISA.
El ensayo se realizó para determinar la máxima dilución de los anticuerpos monoclonales anti-PB2 de Flu provenientes de los hibridomas 1A3E2 y 2F11B1, que permiten la detección del antigeno
viral. Para esto, se activó la placa con 50 ng de antigeno purificado (proteina PB2 ) y luego la placa se bloqueó por 2 horas a 37°C con PBS IX/ Suero Fetal Bovino (FBS) 10%. Los anticuerpos anti-PB2 1A3E2 ó 2F11B1 se utilizaron en diluciones 1:2, partiendo de la concentración de trabajo (170 ng) hasta la dilución 11 (0,15 ng) en PBS 1X/FBS 10%. Posteriormente se adicionó el anticuerpo de detección anti-IgG de ratón en una dilución de 1:2000 (1,8 ng/mΐ por pocilio) se incubo 1 hora a temperatura ambiente ( 25°C), en oscuridad. Finalmente, se realizaron los lavados y se reveló con 50 pL de buffer citrato/Tetrametilbenzidina (TMB, 3-3' -5-5 ' tetrametil- benzidina, lmg/mL, Becton Dickinson) . Para detener la reacción se adicionaron 50 pL de H2SO4 2N y el resultado se leyó en un lector de ELISA, a 450 nm. En la figura 3 se observa que el anticuerpo anti-PB2 1A3E2 puede detectar 50 ng del antigeno purificado hasta con 1,3 ng por pocilio (Figura 3A) . Por otro lado, el clon anti-PB2 2F11B1 es más eficiente que el clon 1A3E2, ya que reconoce 50 ng de PB2 purificada con casi todas las diluciones realizadas (Figura 3B) . El control negativo incluido en este ensayo, corresponde a un pocilio que no contiene muestra (proteina PB2 ) , fue bloqueado con PBS 1X/FBS 10%, se le adicionó anticuerpo primario (anti-PB2 1A3E2 ó anti-PB2 2F11B1) y contiene además anticuerpo anti-IgG de ratón conjugado con HRP . The assay was performed to determine the maximum dilution of Flu's anti-PB2 monoclonal antibodies from 1A3E2 and 2F11B1 hybridomas, which allow detection of antigen viral. For this, the plate was activated with 50 ng of purified antigen (PB2 protein) and then the plate was blocked for 2 hours at 37 ° C with PBS IX / 10% Fetal Bovine Serum (FBS). Anti-PB2 1A3E2 or 2F11B1 antibodies were used in 1: 2 dilutions, starting from the working concentration (170 ng) up to the 11 dilution (0.15 ng) in PBS 1X / 10% FBS. Subsequently, the anti-mouse IgG detection antibody was added at a dilution of 1: 2000 (1.8 ng / mΐ per well) and it was incubated for 1 hour at room temperature (25 ° C), in the dark. Finally, the washes were carried out and it was developed with 50 pL of citrate / Tetramethylbenzidine buffer (TMB, 3-3 '-5-5' tetramethyl-benzidine, lmg / mL, Becton Dickinson). To stop the reaction, 50 pL of H2SO4 2N were added and the result was read in an ELISA reader, at 450 nm. Figure 3 shows that the anti-PB2 antibody 1A3E2 can detect 50 ng of the purified antigen with up to 1.3 ng per well (Figure 3A). On the other hand, the anti-PB2 clone 2F11B1 is more efficient than the clone 1A3E2, since it recognizes 50 ng of purified PB2 with almost all the dilutions made (Figure 3B). The negative control included in this assay corresponds to a well that does not contain a sample (PB2 protein), was blocked with PBS 1X / FBS 10%, primary antibody was added (anti-PB2 1A3E2 or anti-PB2 2F11B1) and also contains HRP-conjugated anti-mouse IgG antibody.
Ejemplo 6: Diagnóstico clínico de muestras de pacientes infectados con Flu, utilizando anticuerpos monoclonales anti- PB2 de Flu, mediante la técnica de ELISA en Sándwich. Example 6: Clinical diagnosis of samples from patients infected with Flu, using Flu anti-PB2 monoclonal antibodies, using the sandwich ELISA technique.
La disponibilidad y la concentración de las proteínas virales generalmente es muy escasa en muestras clínicas de hisopados nasofaríngeos, por lo que fue necesario modificar el ensayo de ELISA realizado previamente. Para este ensayo se realizó un ELISA en Sándwich, usando como anticuerpo de captura el anticuerpo
anti-PB2 proveniente del hibridoma 1A3E2 de Flu y como anticuerpo de detección el clon anti-PB2 2F11B1 de Flu. El anticuerpo de detección anti-PB2 2F11B1 de Flu, se conjugó con HRP. Para el ensayo se activaron pocilios de una placa de ELISA con 3,4 mr/hIII (170 ng/pocillo) del anticuerpo anti-PB2 proveniente del hibridoma 1A3E2 para Flu, el cual se diluyó en PBS IX, y se incubó durante 1 hora a 37 °C. Se realizaron 2 lavados con PBS lX-Tween20 al 0,05% y posteriormente se bloqueó la placa con 200 mE de PBS 1X/FBS al 10% durante 1 hora a 37°C. Se lavó nuevamente y se incubó durante 1 hora a 37°C cada pocilio con 50 mE de hisopados nasofaríngeos (previamente tratados) de pacientes positivos para Flu de acuerdo al método de diagnóstico "D3 Ultra DFA Respiratory Virus Screening and ID Kit de DHI (Diagnostics Hibryds) USA", denominado de manera rutinaria como "panel viral", y que fueron tratadas como se describe posteriormente. Como controles se incluyeron: 1) control de especificidad: se utilizaron 50 mE de muestra de pacientes diagnosticados con PIV mediante el panel viral para los anticuerpos anti-Flu; 2) control positivo: 50 ng de proteina PB2-FÍU recombinante; 3) Control negativo: correspondiente a muestras de controles sanos. Posteriormente, se realizaron los 2 lavados correspondientes con PBS lX-Tween20 0,05% y cada pocilio se incubó por 1 hora a temperatura ambiente con 50 mE del anticuerpo anti-PB2 proveniente del hibridoma 2F11B1 conjugado con HRP (concentración final de 1,8 ng/mΐ por pocilio). Los anticuerpos de detección se incubaron 1 hora a temperatura ambiente ( 25°C) , en oscuridad. A continuación, se lavó la placa 2 veces más, se reveló con 50 mΐ de solución TMB y se incubó durante 15 minutos en oscuridad. La reacción se detuvo con 50 mΐ de H2SO4 2N y la lectura de la placa se realizó a 450 nm en un lector de ELISA (modelo Epoch) , certificado para diagnóstico clínico.
Los resultados obtenidos para este ensayo se muestran en la figura 4A, donde se puede observar que la técnica de ELISA en Sándwich utilizando el anticuerpo (anti-PB2) proveniente del hibridoma 1A3E2, como anticuerpo de captura y el anticuerpo proveniente del hibridoma 2F11B1-HRP como anticuerpo de detección, permite detectar el antigeno en muestras de pacientes infectados con Flu (Figura 4A) , los cuales fueron previamente confirmados por Inmunofluorescencia directa en un laboratorio clinico certificado utilizando el panel viral. La figura 4A, muestra los resultados obtenidos con los anticuerpos anti-PB2 de Flu, donde se utilizaron 12 muestras de pacientes diagnosticados como Flu positivo y como control de especificidad, se incluyeron 3 muestras de pacientes positivos para el virus Parainfluenza. Como control positivo se incluyeron pocilios a los que se añadió proteina recombinante PB2-Flu purificada. Como control negativo se utilizaron 10 controles sanos. Los resultados muestran que los anticuerpos son especificos en detectar sólo los pacientes positivos para Flu y no los controles sanos o los infectados con otro virus (PIV) . Todas las muestras detectadas positivas por ELISA son aquellas que muestran una densidad óptica (OD) por encima de 0,15. The availability and concentration of viral proteins is generally very low in clinical samples of nasopharyngeal swabs, so it was necessary to modify the previously performed ELISA assay. For this assay, a sandwich ELISA was performed, using the antibody as the capture antibody. anti-PB2 from Flu 1A3E2 hybridoma and as detection antibody the anti-PB2 clone 2F11B1 from Flu. Flu's anti-PB2 detection antibody 2F11B1 was conjugated to HRP. For the assay, wells of an ELISA plate were activated with 3.4 mr / hIII (170 ng / well) of the anti-PB2 antibody from the 1A3E2 hybridoma for Flu, which was diluted in PBS IX, and incubated for 1 hour at 37 ° C. 2 washes were performed with 0.05% PBS lX-Tween20 and the plate was subsequently blocked with 200 mE of 1X PBS / 10% FBS for 1 hour at 37 ° C. Each well was washed again and incubated for 1 hour at 37 ° C with 50 mE of nasopharyngeal swabs (previously treated) from Flu-positive patients according to the diagnostic method "D 3 Ultra DFA Respiratory Virus Screening and ID Kit by DHI ( Diagnostics Hibryds) USA ", routinely referred to as" viral panel ", and which were treated as described below. The following were included as controls: 1) specificity control: 50 mE of sample from patients diagnosed with PIV were used through the viral panel for anti-Flu antibodies; 2) positive control: 50 ng of recombinant PB2-FÍU protein; 3) Negative control: corresponding to samples from healthy controls. Subsequently, the 2 corresponding washes were performed with 0.05% PBS lX-Tween20 and each well was incubated for 1 hour at room temperature with 50 mE of the anti-PB2 antibody from the HRF-conjugated 2F11B1 hybridoma (final concentration of 1.8 ng / mΐ per well). Detection antibodies were incubated 1 hour at room temperature (25 ° C), in the dark. The plate was then washed 2 more times, developed with 50 mΐ of TMB solution and incubated for 15 minutes in the dark. The reaction was stopped with 50 mΐ of H2SO4 2N and the plate was read at 450 nm in an ELISA reader (Epoch model), certified for clinical diagnosis. The results obtained for this assay are shown in Figure 4A, where it can be seen that the sandwich ELISA technique using the antibody (anti-PB2) from the 1A3E2 hybridoma, as the capture antibody and the antibody from the 2F11B1-HRP hybridoma As a detection antibody, it allows the antigen to be detected in samples from patients infected with Flu (Figure 4A), which were previously confirmed by direct immunofluorescence in a certified clinical laboratory using the viral panel. Figure 4A shows the results obtained with the Flu anti-PB2 antibodies, where 12 samples from patients diagnosed as Flu positive were used and as a specificity control, 3 samples from patients positive for the Parainfluenza virus were included. As a positive control, wells were included to which purified recombinant PB2-Flu protein was added. As a negative control, 10 healthy controls were used. The results show that the antibodies are specific to detect only Flu-positive patients and not healthy controls or those infected with another virus (PIV). All samples detected positive by ELISA are those showing an optical density (OD) above 0.15.
Este ensayo demuestra la versatilidad que presentan los anticuerpos provenientes de los hibridomas 1A3E2 y 2F11B1 anti- PB2 de Flu, ya que son capases de unirse simultáneamente al antigeno sin competir por el sitio de unión ó interferir entre si. Lo anterior permite la captura y posterior detección de la proteina PB2 en muestras de pacientes. This assay demonstrates the versatility of the antibodies from Flu anti-PB2 hybridomas 1A3E2 and 2F11B1, since they are capable of simultaneously binding to the antigen without competing for the binding site or interfering with each other. This allows the capture and subsequent detection of PB2 protein in patient samples.
Tratamiento de muestras clínicas. Las muestras que se utilizaron para los ensayos fueron obtenidas a partir de hisopados nasofarigeos contenidos en medio de transporte universal (UTM) . Se centrifugaron las muestras a 14.000 rpm durante 4 minutos a temperatura ambiente. Posteriormente se separó el sobrenadante
(SN1) del pellet; este último fue incubado con 100 mύ de Buffer RIPA (50 M Tris-HCl pH 8,0, 150 M NaCl, 1% NP-40, 0,5% Deoxicolato de Sodio, 0,1%, SDS y un coctel de inhibidores de proteasas IX) durante 15 minutos a 4°C, agitando por vortex cada 5 minutos. A continuación, se centrifugó a 14.000 rpm durante 4 minutos a temperatura ambiente. Al finalizar se tomó el sobrenadante obtenido (SN2) y se mezcló con el SN1, se realizó vortex . Treatment of clinical samples. The samples used for the tests were obtained from nasopharyngeal swabs contained in universal transport medium (UTM). Samples were centrifuged at 14,000 rpm for 4 minutes at room temperature. The supernatant was subsequently separated (SN1) of the pellet; the latter was incubated with 100 mύ of RIPA Buffer (50 M Tris-HCl pH 8.0, 150 M NaCl, 1% NP-40, 0.5% Sodium Deoxycholate, 0.1%, SDS and a cocktail of inhibitors of proteases IX) for 15 minutes at 4 ° C, vortexing every 5 minutes. Then, it was spun at 14,000 rpm for 4 minutes at room temperature. At the end, the obtained supernatant (SN2) was taken and mixed with SN1, a vortex was performed.
Es de suma importancia, utilizar ambos anticuerpos para la detección de la proteina PB2, debido a la baja disponibilidad de antigeno en la muestra. Utilizando un ELISA en Sándwich se incrementa la especificidad y sensibilidad en el diagnóstico de Flu. Se realizaron ensayos donde se activó la placa directamente con muestras clinicas de hisopados nasofaríngeos, luego se incubaron los anticuerpos anti-PB2 1A3E2 y anti-PB2 2F11B1, por separado. Luego se incubó un anticuerpo secundario anti-IgG de ratón conjugado con HRP y se evaluó la absorbancia generada al incubar el complejo de anticuerpos más muestra con el sustrato TMB, y no se observó un diagnóstico positivo ya que la señal entregada era muy baja (datos no mostrados) . It is of utmost importance to use both antibodies for the detection of the PB2 protein, due to the low availability of antigen in the sample. Using a sandwich ELISA increases the specificity and sensitivity in the diagnosis of Flu. Assays were performed where the plate was activated directly with clinical samples from nasopharyngeal swabs, then the anti-PB2 1A3E2 and anti-PB2 2F11B1 antibodies were incubated separately. A secondary HRP-conjugated anti-mouse IgG antibody was then incubated and the absorbance generated by incubating the antibody plus sample complex with the TMB substrate was evaluated, and no positive diagnosis was observed as the signal delivered was very low (data not shown).
Realizar un kit diagnóstico utilizando la técnica de ELISA en Sándwich, donde la placa pueda venir activada y bloqueada, disminuirla el tiempo y el costo de realizar diagnóstico, ya que esta técnica es fácil de realizar y analizar en comparación a la técnica estándar (PCR) . El kit no necesita de personal altamente capacitado para realizarlo o analizarlo. Make a diagnostic kit using the sandwich ELISA technique, where the plate can be activated and blocked, reducing the time and cost of making a diagnosis, since this technique is easy to perform and analyze compared to the standard technique (PCR) . The kit does not need highly trained personnel to perform or analyze it.
Ejemplo 7: Diagnóstico clínico de muestras de pacientes infectados con FLU, utilizando anticuerpos monoclonales anti- PB2 de FLU, mediante la técnica de Luminex tipo en Sándwich.
Al igual que en la técnica de ELISA, la disponibilidad y la concentración de las proteínas viral generalmente es muy escasa en muestras clínicas de hisopados nasofaríngeos, por lo que se quiso evaluar por otra técnica más sensible lo obtenido en los resultados por la técnica de ELISA (figura 4A) . Para este ensayo se realizó un ensayo de luminex tipo Sandiwch, usando como anticuerpo de captura el anticuerpo anti-PB2 1A3E2 como anticuerpo de captura y el anti-PB2 2F11B1 como anticuerpo de detección. El anticuerpo de detección anti-PB2 2F11B1 de FLU, se conjugó con el fluorósforo biotina. Placas de Luminex fueron activadas con 50 microesferas magnéticas (marcadas internamente con fluoróforo rojo o cercano al infrarrojo de diferentes intensidades) por mί, las cuales estaban conjugadas con el anticuerpo secretado por el hibridoma 1A3E2 (anti-FLU) , funcionando como anticuerpo de captura (a una concentración final de 2,5 mM) . Las microesferas conjugadas se incubaron con 50 mί de muestras de hisopado nasofaríngeo (HNF) de pacientes que presentaban cuadros respiratorios virales, durante 2 horas a temperatura ambiente (« 23 °C) , en agitación a 400 rpm y en oscuridad (tapados con papel de aluminio) . Como controles negativos se analizaron 8 muestras de controles sanos . Se utilizaron 19 muestras de pacientes positivos para FLU (de acuerdo con el método de diagnóstico "D3 Ultra DFA Respiratory Virus Screening and ID Kit de DHI (Diagnostics Hibryds ) USA", denominado de manera rutinaria como "panel viral", las cuales se trataron de la misma manera mencionada anteriormente, y como control positivo se incluyeron pocilios a los que se añadió proteína PB2-FLU purificada (50 ng) . Pasada las 2 horas, se realizan 2 lavados con 100 mR PBS lX-Tween20 al 0,05% durante 30 segundos utilizando el lavador magnético manual. Para la detección de la proteína capturada por el anticuerpo 1A3E2, se utilizaron los anticuerpos producidos por el hibridoma 2F11B1,
conjugados al fluoróforo biotina, a una concentración de 4 mg/mL diluidos en PBS 1X-BSA 1%, incubándose los pocilios con 50 m]0. La incubación se realiza durante 1 hora a temperatura ambiente, en oscuridad, agitando a 400 rpm. Se realizan nuevamente 2 lavados con 100 mB PBS ÍX-Tween20 al 0,05% durante 30 segundos utilizando el lavador magnético manual. El complejo formado por microesferas conjugadas con anticuerpo de captura más antigeno y anticuerpo de detección, es incubado con 50 mE de Streptavidina/Fi-coeritrina a una concentración final de 6 mr/hϋ. La incubación se realiza durante 30 minutos a temperatura ambiente, en oscuridad, agitando a 400 rpm. Finalmente, se realizan dos pasos de lavados más y se incuban los pocilios con 100 mE del reactivo Sheat fluid (reactivo utilizado por equipo Luminex para que el equipo lea las muestras), agitar 5 minutos a 400 rpm, en oscuridad. Los resultados de la intensidad media de fluorescencia (MFI, por sus siglas en inglés) son entonces lerdos en el equipo Luminex 200, el cual a través de un láser color rojo (621 nm) , detecta la región de reconocimiento de la microesfera y el láser de color verde (511 nm) detecta la unión del anticuerpo de detección al analito . Example 7: Clinical diagnosis of samples from patients infected with FLU, using anti-FLU monoclonal antibodies to FLU, using the Sandwich-type Luminex technique. As in the ELISA technique, the availability and concentration of viral proteins is generally very low in clinical samples of nasopharyngeal swabs, so we wanted to evaluate by another more sensitive technique what was obtained in the results by the ELISA technique. (Figure 4A). For this assay, a Sandiwch-type luminex assay was performed, using the anti-PB2 1A3E2 antibody as the capture antibody and the anti-PB2 2F11B1 as the detection antibody. The FLU anti-PB2 detection antibody 2F11B1 was conjugated to the biotin fluorophore. Luminex plates were activated with 50 magnetic microspheres (internally marked with red or near-infrared fluorophore of different intensities) by mί, which were conjugated with the antibody secreted by the 1A3E2 hybridoma (anti-FLU), functioning as a capture antibody ( at a final concentration of 2.5 mM). The conjugated microspheres were incubated with 50 mί of nasopharyngeal swab (HNF) samples from patients presenting with viral respiratory symptoms, for 2 hours at room temperature ("23 ° C), shaking at 400 rpm and in the dark (covered with tissue paper). aluminum) . As negative controls, 8 samples from healthy controls were analyzed. Nineteen samples of FLU positive patients were used (according to the diagnostic method "D 3 Ultra DFA Respiratory Virus Screening and ID Kit of DHI (Diagnostics Hibryds) USA", routinely referred to as "viral panel", which were treated in the same manner as mentioned above, and as a positive control wells were added to which was added purified PB2-FLU protein (50 ng) After 2 hours, 2 washes were performed with 100 mR PBS lX-Tween20 at 0.05 % for 30 seconds using the manual magnetic washer For the detection of the protein captured by the 1A3E2 antibody, the antibodies produced by the hybridoma 2F11B1 were used, Biotin fluorophore conjugates, at a concentration of 4 mg / mL diluted in PBS 1X-BSA 1%, incubating the wells with 50 m] 0. Incubation is carried out for 1 hour at room temperature, in the dark, shaking at 400 rpm. 2 washes are again performed with 100 mB 0.05% PBS ÍX-Tween20 for 30 seconds using the manual magnetic scrubber. The complex formed by microspheres conjugated with capture antibody plus antigen and detection antibody, is incubated with 50 mE of Streptavidin / Fi-coeritrin at a final concentration of 6 mr / hϋ. Incubation is carried out for 30 minutes at room temperature, in the dark, shaking at 400 rpm. Finally, two more washing steps are carried out and the wells are incubated with 100 mE of the Sheat fluid reagent (reagent used by the Luminex team for the team to read the samples), shake for 5 minutes at 400 rpm, in the dark. The results of the average fluorescence intensity (MFI, for its acronym in English) are then slow on the Luminex 200 equipment, which through a red laser (621 nm), detects the recognition region of the microsphere and the Green laser (511 nm) detects the binding of the detection antibody to the analyte.
Los resultados obtenidos para este ensayo se muestran en la figura 4B, donde se puede observar que la técnica de Luminex, al igual que lo obtenido por la técnica de ELISA, utilizando el anticuerpo (anti-PB2) proveniente del hibridoma 1A3E2, como anticuerpo de captura y el anticuerpo proveniente del hibridoma 2F11B1-HRP como anticuerpo de detección, permite detectar el antigeno en muestras de pacientes infectados con FLU (Figura 4A) con alta intensidad, los cuales fueron previamente confirmados por Inmunofluorescencia directa en un laboratorio clinico certificado utilizando el panel viral. La figura 4B, muestra los resultados obtenidos con los anticuerpos anti-PB2 de FLU, donde
se utilizaron 19 muestras de pacientes diagnosticados como FLU positivo y 6 muestras de controles sanos. Además, se utilizó como control positivo pocilios a los que se añadió proteina PB2- FLU purificada. Los resultados muestran que los anticuerpos anti-PB2 son específicos en detectar sólo los pacientes positivos para FLU y no los sujetos controles. Todas las muestras detectadas como positivas por Luminex son aquellas que muestran un MFI por encima de dos desviaciones estándar del promedio de MFI de los controles sanos. The results obtained for this assay are shown in Figure 4B, where it can be seen that the Luminex technique, like that obtained by the ELISA technique, using the antibody (anti-PB2) from the 1A3E2 hybridoma, as a capture and the antibody from the 2F11B1-HRP hybridoma as a detection antibody, allows the antigen to be detected in samples from patients infected with FLU (Figure 4A) with high intensity, which were previously confirmed by direct immunofluorescence in a certified clinical laboratory using the panel viral. Figure 4B shows the results obtained with the FLU anti-PB2 antibodies, where 19 samples from patients diagnosed as positive FLU and 6 samples from healthy controls were used. In addition, wells to which purified PB2-FLU protein was added were used as a positive control. The results show that anti-PB2 antibodies are specific in detecting only FLU positive patients and not control subjects. All samples detected as positive by Luminex are those showing an MFI above two standard deviations from the average MFI of healthy controls.
Este ensayo, al igual que en el ensayo de ELISA con muestras de pacientes, demuestra la versatilidad que presentan los anticuerpos provenientes de los hibridomas 1A3E2 y 2F11B1 de FLU, ya que son capases de unirse simultáneamente al antigeno sin competir por el sitio de unión o interferir entre si y detectar la poca disponibilidad del antigeno en la muestra de hisopado nasofaríngeo. This assay, as in the ELISA assay with patient samples, demonstrates the versatility of antibodies from FLU 1A3E2 and 2F11B1 hybridomas, since they are capable of simultaneously binding to the antigen without competing for the binding site or interfere with each other and detect the low availability of the antigen in the nasopharyngeal swab sample.
Ejemplo 8: Estudio ciego para la detección del antigeno PB2-FLU en muestras clínicas, obtenidas de pacientes cursando una infección, utilizando anticuerpos monoclonales anti-PB2 de FLU, los cuales forman parte del kit de detección múltiple de virus respiratorios . Example 8: Blind study for the detection of the PB2-FLU antigen in clinical samples, obtained from patients carrying an infection, using anti-FLU monoclonal antibodies to FLU, which are part of the multiple respiratory virus detection kit.
Previamente se realizaron ensayos de ELISA en Sándwich donde se conocía el diagnóstico previo de las muestras a evaluar. Posterior a estos ensayos, se realizó un estudio ciego, donde se evaluaron cerca de 160 muestras de hisopados nasofaríngeos, sin conocer el diagnóstico microbiológico . Para todos los ensayos del estudio ciego, se realizaron ELISA en Sándwich donde se utilizó como anticuerpo de captura el anticuerpo anti-L 1A3E2 y el anti-L 2F11B1 como anticuerpo de detección conjugado con HRP. Para todos los ensayos se activaron pocilios de una placa de
ELISA con 3,4 mg/mL (170 ng/pocillo) del anticuerpo anti-L proveniente del hibridoma 1A3E2 de FLU, diluido en PBS IX, durante 30 minutos a 37°C. Se realizaron 2 lavados con PBS IX- Tween20 al 0,05% y posteriormente se bloqueó la placa con 200 mE de PBS 1X/FBS al 10% durante 30 min a 37°C. Se lavó nuevamente y se incubó durante 1 hora a 37°C cada pocilio con 50 mE de hisopados nasofaríngeos de pacientes, los cuales se evaluaron en paralelo por el método de diagnóstico estándar (PCR), denominado de manera rutinaria como "panel viral", y que fueron tratadas como se describe previamente en el ejemplo 6. Como controles se incluyeron: 1) control de especificidad: se utilizaron 50 mE de la proteina BSA (50 ng) ; 2) control positivo: 50 ng de proteina PB2-FLU recombinante; 3) Controles negativos: pocilios sin muestra y pocilios bloqueados e incubados con anticuerpo de detección. Posteriormente, se realizaron los 2 lavados correspondientes con PBS lX-Tween20 0,05% y cada pocilio se incubó por 30 min a temperatura ambiente ( 25°C, en oscuridad) con 50 mE del anticuerpo anti-PB2 proveniente del hibridoma 2F11B1, conjugado con HRP (1,8 ng/mE de concentración final). A continuación, se lavó la placa 2 veces más, se reveló con 50 mΐ de solución TMB y se incubó durante 15 minutos en oscuridad. La reacción se detuvo con 50 mL de H2SO4 2N y la lectura de la placa se realizó a 450 nm en un lector de ELISA (modelo Epoch) , certificado para diagnóstico clinico. Previously, sandwich ELISA tests were performed where the previous diagnosis of the samples to be evaluated was known. Subsequent to these trials, a blind study was performed, where nearly 160 samples of nasopharyngeal swabs were evaluated, without knowing the microbiological diagnosis. For all blind study assays, Sandwich ELISAs were performed where the anti-L 1A3E2 antibody and the anti-L 2F11B1 were used as the HRP-conjugated detection antibody. For all tests, wells of a plate were activated. ELISA with 3.4 mg / mL (170 ng / well) of the anti-L antibody from the FLU 1A3E2 hybridoma, diluted in PBS IX, for 30 minutes at 37 ° C. 2 washes were performed with 0.05% PBS IX-Tween20 and the plate was subsequently blocked with 200 mE of PBS 1X / 10% FBS for 30 min at 37 ° C. Each well was washed again and incubated for 1 hour at 37 ° C with 50 mE of patient nasopharyngeal swabs, which were evaluated in parallel by the standard diagnostic method (PCR), routinely referred to as "viral panel", and that were treated as previously described in example 6. As controls included: 1) specificity control: 50 mE of the BSA protein (50 ng) were used; 2) positive control: 50 ng of recombinant PB2-FLU protein; 3) Negative controls: wells without sample and wells blocked and incubated with detection antibody. Subsequently, the 2 corresponding washes were performed with PBS lX-Tween20 0.05% and each well was incubated for 30 min at room temperature (25 ° C, in the dark) with 50 mE of the anti-PB2 antibody from the 2F11B1 hybridoma, conjugated with HRP (1.8 ng / mE of final concentration). The plate was then washed 2 more times, developed with 50 mΐ of TMB solution and incubated for 15 minutes in the dark. The reaction was stopped with 50 mL of H2SO4 2N and the plate was read at 450 nm in an ELISA reader (Epoch model), certified for clinical diagnosis.
Los resultados se muestran en la figura 4A, donde se observa la capacidad de los anticuerpos de detectar la proteina PB2 en muestras clinicas, siendo que fueron diseñados a partir de una proteina quimera. Se detectaron 18 de 21 pacientes PIV positivos, y a partir de estos resultados se pudo determinar la exactitud diagnóstica de los anticuerpos, la cual se muestra en la tabla 1. En la tabla se observan los dos conceptos que definen a la
exactitud diagnóstica, donde tenemos la especificidad, es decir la capacidad de los anticuerpos de diagnosticar muestras negativas como negativas, sin detectar falsos positivos, y por otro lado, tenemos la sensibilidad, es decir la capacidad de los anticuerpos de diagnosticar como positivas aquellas muestras que realmente lo son, sin diagnosticar falsos negativos. Los resultados expuestos en la tabla muestran un alto porcentaje de especificidad (94%) y de sensibilidad (86%) de los anticuerpos frente a la técnica estándar (PCR) . The results are shown in figure 4A, where the ability of the antibodies to detect the PB2 protein in clinical samples is observed, since they were designed from a chimera protein. 18 out of 21 positive IVP patients were detected, and from these results the diagnostic accuracy of the antibodies could be determined, which is shown in Table 1. The table shows the two concepts that define the diagnostic accuracy, where we have specificity, that is, the ability of antibodies to diagnose negative as negative samples, without detecting false positives, and on the other hand, we have sensitivity, that is, the ability of antibodies to diagnose as positive those samples that They really are, without diagnosing false negatives. The results shown in the table show a high percentage of specificity (94%) and sensitivity (86%) of the antibodies against the standard technique (PCR).
Tabla 1. Exactitud diagnóstica de los anticuerpos anti-PB2-FLU Table 1. Diagnostic Accuracy of Anti-PB2-FLU Antibodies.
Ejemplo 9: Detección de proteina PB2 mediante ensayo de ELISA indirecto, utilizando los anticuerpos monoclonales completos y fragmentos de ellos . Example 9: Detection of PB2 protein by indirect ELISA assay, using whole monoclonal antibodies and fragments of them.
En este ejemplo de aplicación se demuestra que tanto el anticuerpo monoclonal especifico contra la proteina PB2 puede ser detectado mediante ELISA indirecto. Para esto, placas de ELISA fueron activadas con 50 mE de de 50 ng de proteina PB2 y BSA. Los sitios inespecificos fueron bloqueados con FBS al 10% diluido en PBS IX. 170 ng (3,4 mg/mL) de los fragmentos Fab de los anticuerpos secretados por el hibridoma 1A3E2 (anti-Flu) y 2F11B1 (anti-Flu), ambos previamente conjugados biotina. Incubación de moléculas de unión a biotina (Estreptavidina) , la cual está conjugada con HRP (dilución 1:2000, 75 ng por pocilio)
(Figura 5, barra de color gris oscuro, anticuerpo 1A3E2 y barra de color gris claro, anticuerpo 2F11B1) . In this application example it is shown that both the specific monoclonal antibody against the PB2 protein can be detected by indirect ELISA. For this, ELISA plates were activated with 50 mE of 50 ng of PB2 protein and BSA. Non-specific sites were blocked with 10% FBS diluted in PBS IX. 170 ng (3.4 mg / mL) of the Fab fragments of the antibodies secreted by the hybridoma 1A3E2 (anti-Flu) and 2F11B1 (anti-Flu), both previously conjugated biotin. Incubation of biotin-binding molecules (Streptavidin), which is conjugated to HRP (1: 2000 dilution, 75 ng per well) (Figure 5, dark gray bar, 1A3E2 antibody and light gray bar, 2F11B1 antibody).
Ejemplo 10: Ensayo de detección de antigenos de Flu, utilizando fragmentos F(ab' )2 de los anticuerpos monoclonales anti-PB2 de Flu mediante ensayo de ELISA indirecto Example 10: Flu antigen detection assay, using F (ab ') 2 fragments of Flu anti-PB2 monoclonal antibodies by indirect ELISA assay
Este ensayo tiene como objetivo demostrar la capacidad de detección de fragmentos de los anticuerpos anti-Flu, producidos por los hibridomas 1A3E2 y 2F11B1, por la proteina PB2. Previamente al ensayo de ELISA indirecto, se realizó la fragmentación de la molécula de IgG de cada anticuerpo anti Flu. La fragmentación se realizó utilizando el kit "Thermo Scientific™ Kits de preparación de fragmentos F(ab' )2 Pierce™" (#10381214, Thermo Scientific), el cual separa el fragmento F(ab' )2 y el Fe del anticuerpo de interés, mediante el uso de la enzima pepsina que digiere el fragmento Fe y posteriormente se realizan pasos de purificación para separar el fragmento F(ab' )2 del fragmento Fe digerido. Posterior a la fragmentación del anticuerpo, la fracción F(ab' )2 purificada fue comprobada por la técnica de western blot. Fracciones F(ab' )2 fueron conjugadas con moléculas de biotina utilizando el kit de conjugación rápida, Lightning-Link rapid biotin type A (#370-0010, Expedeon) . Teniendo todos los reactivos listos, se llevó a cabo la detección del antigeno mediante la técnica de ELISA indirecto, donde la placa de ELISA se activó con 50 ng de antigeno PB2 purificado por 1 hora a 37°C. Se incluyeron dos controles negativos, uno sin muestra y otro incubando el pocilio con 50 ng de proteina BSA. Posteriormente, la placa se lavó dos veces con buffer fosfato salino (PBS) lX/Tween20 0,05%. Luego la placa se bloqueó por 2 horas a 37 °C con PBS IX/ Suero Fetal Bovino (FBS) 10%. Posteriormente se repitieron los lavados y a continuación se incubaron cada uno de los anticuerpos, sin fraccionar y
fracciones F(ab')2 (1A3E2 y 2F11B1) conjugados con biotina, a una concentración final de 3,4 mg/mL (170 ng por pocilio), diluidos en PBS 1X/FBS 10%, por 1 hora a 37°C (cada anticuerpo en una placa independiente) . Transcurrido el tiempo de incubación, se repitieron los lavados y se agregó a cada uno de los pocilios con una proteina de unión a biotina (Estreptavidina) marcada con la enzima peroxidasa de rábano (Horseradish peroxidase, HRP) en dilución 1 en 2000 (25 ng/mE por pocilio) en PBS 1X/FBS 10%, por 1 hora a temperatura ambiente ( 25°C) , en oscuridad. Finalmente, se realizaron los lavados y se reveló con 50 L de buffer citrato/Tetrametilbenzidina (TMB, 3-3' -5- 5 ' tetramethylbenzidine , lmg/ml, Becton Dickinson) . Para detener la reacción se adicionaron 50 pL de H2SO4 2N y el resultado se leyó en un lector de ELISA, a 450nm. Para determinar que la reacción del anticuerpo secundario era especifica en reconocer al anticuerpo primario y además que la señal obtenida no sea provocada por unión inespecifica del anticuerpo secundario al antigeno, se realizaron controles en los cuales se utilizó solamente el anticuerpo secundario sin anticuerpo primario ni muestra (pocilio sin activar) . Otro control para determinar que la reacción del anticuerpo primario es especifica para el antigeno, consistió en el uso de los anticuerpos sobre una placa de ELISA que no ha sido activada con el antigeno (sin muestra) o usando los anticuerpos sobre una placa de ELISA que poseía 50 ng de la proteina BSA. Los resultados muestran que los anticuerpos monoclonales de la invención son capaces de reconocer 50 ng de antigeno purificado, de manera especifica, independiente de que se utilice el anticuerpo completo o un fragmento del mismo (Figura 5, barra de color negro, anticuerpo 1A3E2 y barra de color blanco, anticuerpo 2F11B1) .
This assay aims to demonstrate the ability to detect fragments of anti-Flu antibodies, produced by hybridomas 1A3E2 and 2F11B1, by protein PB2. Prior to the indirect ELISA assay, the IgG molecule of each anti-Flu antibody was fragmented. Fragmentation was performed using the "Thermo Scientific ™ F (ab ') 2 Pierce ™ Fragment Preparation Kits" kit (# 10381214, Thermo Scientific), which separates the F (ab') 2 fragment and Fe from the Of interest, by using the enzyme pepsin that digests the Fe fragment and subsequently purification steps are performed to separate the F (ab ') 2 fragment from the digested Fe fragment. After fragmentation of the antibody, the purified F (ab ') 2 fraction was verified by the western blot technique. F (ab ') 2 fractions were conjugated to biotin molecules using the Lightning-Link rapid biotin type A rapid conjugation kit (# 370-0010, Expedeon). Having all the reagents ready, the detection of the antigen was carried out using the indirect ELISA technique, where the ELISA plate was activated with 50 ng of purified PB2 antigen for 1 hour at 37 ° C. Two negative controls were included, one without a sample and the other incubating the well with 50 ng of BSA protein. Subsequently, the plate was washed twice with phosphate buffered saline (PBS) lX / Tween20 0.05%. The plate was then blocked for 2 hours at 37 ° C with PBS IX / 10% Fetal Bovine Serum (FBS). Subsequently, the washes were repeated and each of the antibodies was then incubated, without fractionating and biotin-conjugated F (ab ') 2 fractions (1A3E2 and 2F11B1), at a final concentration of 3.4 mg / mL (170 ng per well), diluted in PBS 1X / FBS 10%, for 1 hr at 37 ° C (each antibody on a separate plate). At the end of the incubation time, the washes were repeated and a biotin-binding protein (Streptavidin) labeled with the horseradish peroxidase enzyme (Horseradish peroxidase, HRP) in dilution 1 in 2000 (25 ng) was added to each well. / mE per well) in PBS 1X / FBS 10%, for 1 hour at room temperature (25 ° C), in the dark. Finally, the washes were performed and revealed with 50 L of citrate / Tetramethylbenzidine buffer (TMB, 3-3 '-5- 5' tetramethylbenzidine, lmg / ml, Becton Dickinson). To stop the reaction, 50 pL of H2SO4 2N were added and the result was read in an ELISA reader, at 450nm. To determine that the reaction of the secondary antibody was specific to recognize the primary antibody and also that the signal obtained was not caused by nonspecific binding of the secondary antibody to the antigen, controls were performed in which only the secondary antibody was used without primary antibody or sample. (well not activated). Another control to determine that the reaction of the primary antibody is specific for the antigen, consisted of using the antibodies on an ELISA plate that has not been activated with the antigen (without sample) or using the antibodies on an ELISA plate that he had 50 ng of the BSA protein. The results show that the monoclonal antibodies of the invention are able to recognize 50 ng of purified antigen, specifically, regardless of whether the whole antibody or a fragment thereof is used (Figure 5, black bar, 1A3E2 antibody and bar white, antibody 2F11B1).
Claims
1. Anticuerpo monoclonal o una porción de unión a antigeno del mismo que se une a la proteina PB2 del virus de la influenza humana (FLU) para su uso en la detección de la presencia y/o localización de la proteina CARACTERIZADO porque el anticuerpo se selecciona de: 1. Monoclonal antibody or an antigen-binding portion thereof that binds to the human influenza virus (FLU) PB2 protein for use in detecting the presence and / or localization of the protein CHARACTERIZED because the antibody is select from:
i) un anticuerpo que comprende una región variable de cadena ligera donde su CDR1 (CDRLCI) se define según la SEQ ID NO: 1, sui) an antibody comprising a light chain variable region where its CDR1 (CDR LCI ) is defined according to SEQ ID NO: 1, its
CDR2 (CDRLC2 ) se define por la SEQ ID NO: 2 y su CDR3 (CDRLCS) corresponde a la SEQ ID NO: 3, y una región variable de la cadena pesada donde su CDR1 (CDRHCI) se define según la SEQ ID NO: 4, su CDR2 (CDRHC2 ) corresponde a la SEQ ID NO: 5 y su CDR3 (CDRHCS) corresponde a la SEQ ID NO: 6, o CDR2 (CDR LC2) is defined by SEQ ID NO: 2 and its CDR3 (CDR LCS ) corresponds to SEQ ID NO: 3, and a variable region of the heavy chain where its CDR1 (CDR HCI ) is defined according to SEQ ID NO: 4, your CDR2 (CDR HC2) corresponds to SEQ ID NO: 5 and your CDR3 (CDR HCS ) corresponds to SEQ ID NO: 6, or
ii) un anticuerpo que comprende una región variable de cadena ligera donde su CDR1 (CDRLCI) se define según la SEQ ID NO: 7, suii) an antibody comprising a light chain variable region where its CDR1 (CDR LCI ) is defined according to SEQ ID NO: 7, its
CDR2 ( CDRLC2 ) se define por la SEQ ID NO: 8 y su CDR3 (CDRLCS) corresponde a la SEQ ID NO: 9, y una región variable de la cadena pesada donde su CDR1 (CDRHCI) se define según la SEQ ID NO: 10, su CDR2 (CDRHCS) corresponde a la SEQ ID NO: 11 y su CDR3 (CDRHCS) corresponde a la SEQ ID NO: 12. CDR2 (CDR LC 2) is defined by SEQ ID NO: 8 and its CDR3 (CDR LCS ) corresponds to SEQ ID NO: 9, and a variable region of the heavy chain where its CDR1 (CDR HCI ) is defined according to SEQ ID NO: 10, your CDR2 (CDR HCS ) corresponds to SEQ ID NO: 11 and your CDR3 (CDR HCS ) corresponds to SEQ ID NO: 12.
Donde, el anticuerpo puede ser usado como anticuerpo de detección o anticuerpo de captura. Wherein, the antibody can be used as the detection antibody or capture antibody.
2. Método para detectar al virus Flu en una muestra biológica CARACTERIZADO porque el método comprende poner en contacto la muestra biológica con el anticuerpo monoclonal o una porción de unión a antigeno del mismo que se une a la proteina PB2 de Flu de acuerdo a la reivindicación 1 y detectar la unión del anticuerpo con el antigeno, detectando asi el virus Flu en la muestra .
2. Method for detecting Flu virus in a biological sample CHARACTERIZED because the method comprises contacting the biological sample with the monoclonal antibody or an antigen binding portion thereof that binds to the Flu PB2 protein according to claim 1 and detect the binding of the antibody to the antigen, thus detecting the Flu virus in the sample.
3. Método para detectar al virus Flu en una muestra biológica de acuerdo a la reivindicación 2, CARACTERIZADO porque la muestra biológica se selecciona del grupo compuesto por células ín vítro infectadas con Flu, secreciones nasales, lavados nasales, liquido cefalorraquideo , secreciones faríngeas y/o lavados o secreciones bronquiales. 3. Method for detecting Flu virus in a biological sample according to claim 2, CHARACTERIZED in that the biological sample is selected from the group consisting of flu-infected in vitro cells, nasal secretions, nasal lavages, cerebrospinal fluid, pharyngeal secretions and / or washes or bronchial secretions.
4. Método para detectar al virus Flu en una muestra biológica de acuerdo a la reivindicación 2 de acuerdo a cualquiera de las reivindicaciones 2 o 3, CARACTERIZADO porque el ensayo utilizado para la detección de la unión del anticuerpo con el antigeno se selecciona de: ELISA, inmunofluorescencia, inmunohistoquimica, inmunocromatografia, citometria de flujo, cell sorter, inmunoprecipitación y/o Western blot . 4. Method for detecting Flu virus in a biological sample according to claim 2 according to any of claims 2 or 3, CHARACTERIZED because the assay used to detect the binding of the antibody to the antigen is selected from: ELISA , immunofluorescence, immunohistochemistry, immunochromatography, flow cytometry, cell sorter, immunoprecipitation and / or Western blot.
5. Método para detectar al virus Flu en una muestra biológica de acuerdo a cualquiera de las reivindicaciones 2 a 4, 5. Method for detecting Flu virus in a biological sample according to any of claims 2 to 4,
CARACTERIZADO porque el anticuerpo o una porción de unión a antigeno del mismo de acuerdo con la reivindicación 1, se encuentra conjugado con un marcador que permite su detección.CHARACTERIZED in that the antibody or an antigen binding portion thereof according to claim 1, is conjugated with a marker that allows its detection.
6. Método para detectar al virus Flu en una muestra biológica de acuerdo a la reivindicación 5, CARACTERIZADO porque el anticuerpo se encuentra unido a un marcador seleccionado del grupo compuesto por fluoróforos, biotina, radioisótopos, metales y enzimas. 6. Method for detecting the Flu virus in a biological sample according to claim 5, CHARACTERIZED because the antibody is linked to a marker selected from the group consisting of fluorophores, biotin, radioisotopes, metals and enzymes.
7. Kit para la detección cualitativa y/o cuantitativa de virus Flu CARACTERIZADO porque comprende:
un anticuerpo monoclonal o una porción de unión a antigeno del mismo que se une a la proteina quimérica L de PIV de acuerdo a la reivindicación 1, el que actúa como anticuerpo de captura o de detección, donde el anticuerpo de detección se encuentra conjugado a un marcador para su detección; un soporte sólido al cual se adosa el anticuerpo; y reactivos para detectar el marcador incluido en el anticuerpo de detección, tales como fluoróforos, biotina, radioisótopos, metales y enzimas. 7. Kit for the qualitative and / or quantitative detection of Flu virus, CHARACTERIZED because it includes: a monoclonal antibody or an antigen-binding portion thereof that binds to the PIV chimeric protein L according to claim 1, which acts as a capture or detection antibody, wherein the detection antibody is conjugated to a marker for detection; a solid support to which the antibody is attached; and reagents for detecting the marker included in the detection antibody, such as fluorophores, biotin, radioisotopes, metals, and enzymes.
8. Kit para la detección cualitativa y/o cuantitativa de virus8. Kit for qualitative and / or quantitative virus detection
Flu de acuerdo con la reivindicación 7, CARACTERIZADO porque el soporte sólido es una membrana formada por uno de los compuestos seleccionados del grupo compuesto por nitrocelulosa, celulosa, polietileno y nylon. Flu according to claim 7, CHARACTERIZED in that the solid support is a membrane formed by one of the compounds selected from the group consisting of nitrocellulose, cellulose, polyethylene and nylon.
9. Kit para la detección cualitativa y/o cuantitativa de virus9. Kit for qualitative and / or quantitative virus detection
Flu de acuerdo a cualquiera de las reivindicaciones 7 y 8, CARACTERIZADO porque corresponde a un test inmunocromatográfico , luminex, citometria de flujo, inmunofluorescencia, radioinmunoanálisis , Western blot, Dot plot, ELISA, inmunodifusión o inmunoprecipitación, para detectar PIV. Flu according to any of claims 7 and 8, CHARACTERIZED because it corresponds to an immunochromatographic test, luminex, flow cytometry, immunofluorescence, radioimmunoassay, Western blot, Dot plot, ELISA, immunodiffusion or immunoprecipitation, to detect PIV.
10. Uso de un anticuerpo monoclonal o una porción de unión a antigeno del mismo que se une a la proteina PB2 de FLU de acuerdo con la reivindicación 1 CARACTERIZADO porque sirve como anticuerpo de captura y/o detección.
10. Use of a monoclonal antibody or an antigen-binding portion thereof that binds to the FLU PB2 protein according to claim 1 CHARACTERIZED in that it serves as a capture and / or detection antibody.
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| CN201980093160.6A CN113490684A (en) | 2018-12-28 | 2019-12-27 | Monoclonal antibodies specific for the PB2 antigen of the human influenza virus (FLU), nucleotide sequences, methods and kits for diagnosing infections produced by FLU |
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| PE2021001088A PE20211697A1 (en) | 2018-12-28 | 2019-12-27 | MONOCLONAL ANTIBODIES SPECIFIC TO HUMAN INFLUENZA VIRUS (FLU) PB2 ANTIGEN, NUCLEOTIDE SEQUENCES; FLU INFECTION DIAGNOSTIC KIT AND METHOD |
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006051069A2 (en) * | 2004-11-11 | 2006-05-18 | Solvay Pharmaceuticals B.V. | Defective influenza virus particles |
| WO2012045001A2 (en) | 2010-09-30 | 2012-04-05 | Vanderbilt University | Influenza virus antibodies and immunogens and uses therefor |
| JP2013540701A (en) * | 2010-08-12 | 2013-11-07 | セラクローン サイエンシーズ， インコーポレイテッド | Anti-hemagglutinin antibody composition and method of use thereof |
| AU2008309939B2 (en) * | 2007-10-09 | 2013-11-14 | European Molecular Biology Laboratory (Embl) | Soluble fragments of influenza virus PB2 protein capable of binding RNA-cap |
| JP2015189715A (en) | 2014-03-28 | 2015-11-02 | 公益財団法人神奈川科学技術アカデミー | Monoclonal antibodies against pb2 subunit of rna-dependent rna polymerase of influenza virus |
| US9650434B2 (en) | 2013-04-02 | 2017-05-16 | Xiamen University | Broad-spectrum monoclonal antibody recognizing HA1 domain of hemagglutinin of influenza virus |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016047592A1 (en) * | 2014-09-22 | 2016-03-31 | 国立研究開発法人科学技術振興機構 | Anti-influenza virus agent, and screening method for anti-influenza virus agent |
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Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006051069A2 (en) * | 2004-11-11 | 2006-05-18 | Solvay Pharmaceuticals B.V. | Defective influenza virus particles |
| AU2008309939B2 (en) * | 2007-10-09 | 2013-11-14 | European Molecular Biology Laboratory (Embl) | Soluble fragments of influenza virus PB2 protein capable of binding RNA-cap |
| JP2013540701A (en) * | 2010-08-12 | 2013-11-07 | セラクローン サイエンシーズ， インコーポレイテッド | Anti-hemagglutinin antibody composition and method of use thereof |
| WO2012045001A2 (en) | 2010-09-30 | 2012-04-05 | Vanderbilt University | Influenza virus antibodies and immunogens and uses therefor |
| US9650434B2 (en) | 2013-04-02 | 2017-05-16 | Xiamen University | Broad-spectrum monoclonal antibody recognizing HA1 domain of hemagglutinin of influenza virus |
| JP2015189715A (en) | 2014-03-28 | 2015-11-02 | 公益財団法人神奈川科学技術アカデミー | Monoclonal antibodies against pb2 subunit of rna-dependent rna polymerase of influenza virus |
Non-Patent Citations (5)
| Title |
|---|
| "Anti PB2 [Clone# 3-1.6]", CATALOG NO. KAST-MA001, KAST-MA003, July 2018 (2018-07-01), pages 1 - 2, XP055829003, Retrieved from the Internet <URL:https://search.cosmobio.co.jp/cosmo_search_p/search_gate2/docs/CAC_/KASTMA003.20160913.pdf> [retrieved on 20200331] * |
| CATALOGO DE PRODUCTOS COSMO BIO NEWS, July 2018 (2018-07-01), XP055723437, Retrieved from the Internet <URL:https://www.cosmobio.co.jp/upfiles/catalog/pdf/catalog_12890.pdf> [retrieved on 20200331] * |
| CHAISRI, U.: "Evolution of therapeutic antibodies, influenza virus biology, influenza, and influenza immunotherapy", BIOMED RESEARCH INTERNATIONAL, vol. 2018, 2018, XP055723440 * |
| HATTA, M. ET AL.: "Mapping of functional domains on the influenza A virus RNA polymerase PB2 molecule using monoclonal antibodies", ARCHIVES OF VIROLOGY, vol. 145, no. 9, 2000, pages 1947 - 1961, XP035411136, DOI: 10.1007/s007050070068 * |
| See also references of EP3904380A4 |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115057925A (en) * | 2022-06-22 | 2022-09-16 | 中国检验检疫科学研究院 | Anti-akabane virus monoclonal antibody and application thereof |
| CN115057925B (en) * | 2022-06-22 | 2024-03-26 | 中国检验检疫科学研究院 | Anti-akabane virus monoclonal antibody and application thereof |
Also Published As
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| PE20211697A1 (en) | 2021-09-01 |
| CN113490684A (en) | 2021-10-08 |
| BR112021012792A2 (en) | 2021-11-16 |
| KR20210110662A (en) | 2021-09-08 |
| UY38533A (en) | 2020-07-31 |
| MX2021007871A (en) | 2021-12-10 |
| ZA202104989B (en) | 2023-01-25 |
| US20220119503A1 (en) | 2022-04-21 |
| AR117546A1 (en) | 2021-08-11 |
| CO2021008774A2 (en) | 2021-07-19 |
| EP3904380A4 (en) | 2022-05-11 |
| CA3125206A1 (en) | 2020-07-02 |
| EP3904380A1 (en) | 2021-11-03 |
| CL2018003871A1 (en) | 2021-01-15 |
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